Peripheral gene expression biomarkers for autism

ABSTRACT

The disclosed invention comprises methods and materials for screening cells for genetic profiles associated with autism spectrum disorders. The methods typically involve isolating a cell from an individual and then observing the expression profile of one or more genes in the cell, wherein certain expression patterns of the genes observed are associated with autism spectrum disorders.

REFERENCE TO RELATED APPLICATIONS

This application claims priority under Section 119(e) from U.S.Provisional Application Ser. No. 61/053,316, filed May 15, 2008, thecontents of which are incorporated herein by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with Government support of Grant No. MH064547,awarded by the National Institutes of Health. The Government has certainrights on this invention.

FIELD OF THE INVENTION

The invention relates to methods and materials for observing geneexpression profiles that are associated with conditions such as autism.

BACKGROUND OF THE INVENTION

Autism comprises a behaviorally defined spectrum of disorderscharacterized by impairment of social interaction, deficiency orabnormality of speech development, and limited activities and interest.To standardize the diagnosis of autism spectrum disorders (ASD),diagnostic criteria have been defined by the World Health Organization(International Classification of Diseases, 10th Revision (ICD-10), 1992)and the American Psychiatric Association (Diagnostic and StatisticalManual of Mental Disorders, 4th edition, Text Revision. Washington DC,American Psychiatric Association, 2000 (DSM-IV)).

Genetic factors are significant determinants of autism spectrumdisorders (see, e.g. Geschwind et al., (2007), Curr Opin Neurobiol, 17,103-11). It has been shown that individuals with ASD carry chromosomalabnormality at a greater frequency than the general population (see,e.g. Veenstra-Vanderweele et al., (2004), Annu Rev Genomics Hum Genet,5, 379-405; Vorstman et al., (2006), Mol Psychiatry, 11, 1, 18-28;Jacquemont et al. (2006), J Med Genet, 43, 843-9; Szatmari et al.(2007), Nat Genet, 39, 319-28; Sebat et al. (2007), Science, 316,445-9). Maternally inherited duplication of 15q11-13 (dup15q) is themost common chromosomal abnormality in ASD. Over-expression of geneslocated in the duplicated region, including cytoplasmic FMR1 interactingprotein 1 (CYFIP1), was shown in lymphoblastoid cell lines from ASD withdup15q (see, e.g. Nishimura et al. (2007), Hum Mol Genet, 16, 1682-98).A cryptic deletion at the boundary of the first exon and first intron ofataxin-2 binding protein-1 (A2BP1) was identified in a female with ASD,resulting reduced mRNA expression in the individual's lymphocytes (see,e.g. Martin et al. (2007), Am J Med Genet B Neuropsychiatr Genet). Lossof copy number of neurexin 1 (NRXN1) was identified in two females sibswith ASD but not in either parent (see, e.g. Szatmari et al. (2007), NatGenet, 39, 319-28). Loss of copy number and decreased expression of SH3and multiple ankyrin repeat domains 3 (SHANK3) were identified in fourindividuals with ASD (see, e.g. Jeffries et al., (2005), Am J Med GenetA, 137, 139-47; and Durand et al. (2007), Nat Genet, 39, 25-7).Recently, a common ‘C’ allele in the promoter region of metprotooncogene (MET) was shown to have strong association with ASD (see,e.g. Campbell et al., (2006), Proc Natl Acad Sci USA, 103, 16834-9). The‘C’ variant causes a twofold decrease in MET promoter activity. Thesefindings suggest that dysregulation of gene expression due to variationin genomic sequence may affect susceptibility or cause ASD.

Transcriptome profiling using DNA microarray represents an efficientmanner in which to uncover unanticipated relationship between geneexpression alterations and neuropsychiatric diseases (see, e.g.Geschwind, D. H. (2003), Lancet Neurol, 2, 275-82; and Mimics et al.,(2006), Biol Psychiatry, 60, 163-76). Several studies have suggestedthat blood-derived cells can be used to identify candidate genes inneuropsychiatric diseases, including ASD. Hu et al. analyzed geneexpression profiling of lymphoblastoid cells from monozygotic (MZ) twinsdiscordant in severity of ASD (see, e.g. Hu et al., (2006), BMCGenomics, 7, 118). Several genes were differentially expressed betweenMZ twins, suggesting candidate genes for ASD may be differentiallyexpressed in lymphoblastoid cells from individuals with ASD. Wepreviously analyzed genome-wide expression profiles of lymphoblastoidcells from ASD with full mutation of FMR1 (FMR1-FM) or dup15q, each ofwhich account for 1-2% of ASD cases in large series, and non-autisticcontrols (see, e.g. Nishimura et al. (2007), Hum Mol Genet, 16,1682-98). The gene expression profiles clearly distinguished ASD fromcontrols and separated individuals with ASD based on their geneticetiology. The expression profiles also revealed shared pathways betweenASD with FMR1-FM and ASD with dup15q.

While progress in understanding genetic factors associated with autismspectrum disorders has been made, specific assays for constellations ofgenetic factors associated with autism spectrum disorders would be asignificant benefit to medical personnel. Tests for genetic factorsassociated with autism spectrum disorders are valuable for the diagnosisof this syndrome, as well as useful for research on the geneticmechanisms involved in autism spectrum disorders. Moreover, while thereis no known medical treatment for autism, success has been reported forearly intervention with behavioral therapies. In this context, an assaywould facilitate the early identification of the disease, one nowtypically diagnosed between ages three and five. Thus, there is a needfor methods and materials that can be used to identify subjects havinggenetic polymorphisms associated with autism spectrum disorders.

SUMMARY OF THE INVENTION

Autism spectrum disorder is a heterogeneous condition and is likely toresult from the combined effects of multiple, subtle genetic changesinteracting with environmental factors. The disclosure provided hereinshows that genome-wide expression profiling of lymphoblastoid cells fromASD subjects distinguishes different forms of ASD and reveals sharedpathways. This disclosure identifies genes dysregulated in common amongthe idiopathic ASD as well as ASD with known genetic disorders. Theseresults provide evidence that studies of gene expression in cells suchas blood derived lymphoblastoid cells can be used for example in assaysdesigned to identify and characterize specific polymorphisms associatedwith ASD.

The invention disclosed herein has a number of embodiments. Oneillustrative embodiment is a method of identifying a human cell having agene expression profile associated with autism spectrum disorderscomprising: observing an expression profile of at least one gene in thecell whose expression is shown to be dysregulated in autism spectrumdisorders (e.g. one or more of the genes disclosed in the Tables below);wherein an expression profile of this gene that is at least two, threeor four standard deviations from a mean expression profile of the genein a control cell identifies the human cell as having a gene expressionprofile associated with autism spectrum disorders. Typically, suchmethods are used to facilitate the diagnosis of an autism spectrumdisorder. For example, in certain embodiments of the invention, the cellexamined by this method is obtained from an individual identified asbeing predisposed to and/or exhibiting a behavior associated with autismspectrum disorders, while the control cell is one obtained from anindividual previously identified as not being predisposed to and/orexhibiting a behavior associated with autism spectrum disorders. Incertain embodiments, the cell examined by this method and the controlcell are obtained from individuals who are related as siblings or as aparent and a child. Typically one or more cells used in these methodsare leukocytes obtained from the peripheral blood.

In illustrative methods for observing an expression profile of one ormore genes, mRNA expression is observed, for example by using a usingquantitative PCR (qPCR) technique. In certain embodiments of theinvention, the expression profile of the genes in is observed using amicroarray of polynucleotides. Alternatively, polypeptide expression isobserved and quantified, for example by using an antibody specific for apolypeptide encoded by a gene whose expression is shown to bedysregulated in autism spectrum disorders (e.g. using an ELISA techniqueor the like). Alternatively, the expression profile of a gene isobserved using a Southern blotting technique (e.g. to identify deletionsand/or duplications in genomic sequences).

Embodiments of the invention include kits comprising, for example, afirst container, a label on said container, and a composition containedwithin said container; wherein the composition includes polymerase chainreaction (PCR) primer effective in the quantitative real time analysisof the mRNA expression levels of one or more genes disclosed hereinwhose expression is shown to be dysregulated in autism spectrumdisorders (e.g. one or more of the genes disclosed in the Tables below);the label on said container, or a package insert included in saidcontainer indicates that the composition can be used to observeexpression levels of these genes in at least one type of humanleukocyte; a second container comprising a pharmaceutically-acceptablebuffer; and instructions for using the PCR primer to obtain anexpression profile of the one or more genes. Optionally the kitcomprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 polymerase chain reaction (PCR)primers effective in the quantitative real time analysis of the mRNAexpression levels of different genes disclosed in the Tables below.

In some embodiments of the invention, one can observe an expressionprofile of at least, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more genes whoseexpression is shown to be dysregulated in autism spectrum disorders(e.g. using microarray technologies). In certain embodiments of theinvention, the method is performed on a plurality of individuals and theresults are then categorized based upon similarities or differences intheir gene expression profiles. Optionally, the expression profile(s) isobserved and/or collected and/or stored using a computer systemcomprising a processor element and a memory storage element adapted toprocess and store data from one or more expression profiles (e.g. in alibrary of such profiles). In this context, certain embodiments of theinvention comprise an electronically searchable library of profiles,wherein the profiles include an individual's gene expression data incombination with other diagnostic data, for example assessments ofbehavior associated with autism spectrum disorders.

Other objects, features and advantages of the present invention willbecome apparent to those skilled in the art from the following detaileddescription. It is to be understood, however, that the detaileddescription and specific examples, while indicating some embodiments ofthe present invention are given by way of illustration and notlimitation. Many changes and modifications within the scope of thepresent invention may be made without departing from the spirit thereof,and the invention includes all such modifications.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides data showing that hierarchical clustering and principalcomponent analysis differentiate individuals based on their etiology.ANOVA identified 293 probes with significantly different expressionbetween autism with FMR1-FM (n=8), autism with dup(15q) (n=7) andcontrol (n=15). The probes were subjected to hierarchical clustering andprincipal component analysis (PCA). A) Hierarchical clustering of the 30individuals and genes. Each row represents an individual and each columnrepresent one of the 293 probes. A pseudo-colored representation of therelative intensity was shown, such that a red color indicates highexpression and green color low expression, with the scale shown below.Relative distance of each probe (horizontal axis) and individuals(vertical axis) are also demonstrated. B) Enlargement of thehierarchical clustering dendrogram of the sample in A). All 8 autismwith FMR1-FM, 7 autism with dup(15q), and 15 controls correctlyclustered within their etiological categories. The scale showed theSpearman rank correlation coefficient used to construct the dendrogram.C) PCA of the expression profile of the 293 probes from 30 individuals.Shown here were three principle components. Autism with FMR1-FM weredepicted as red, autism with dup(15q) as green and control as blue. Theindividuals were clustered closely according to their geneticetiologies.

FIG. 2 shows differentially expressed probes identified by threedifferent statistical methods, ANOVA, SAM and RankProd. Venn diagramshowing the number of probes identified as differentially expressedbetween A) autism with FMR1-FM (n=8) and control (n=15) and B) autismwith dup(15q) (n=7) and control (n=15). C) Overlap of the differentiallyexpressed probes (genes) in autism with FMR1-FM and dup(15q).

FIG. 3 shows a confirmation of the differential gene expression byqRTPCR. Total RNA was extracted from lymphoblastoid cells with FMR1-FM,dup(15q) or control and qRTPCR were performed to confirm thedifferential expression identified by microarray analysis. A) Genesspecifically dysregulated in autism with FMR1-FM or dup(15q). B) Genescommonly up-regulated in autism with FMR1-FM and dup(15q). C) Genescommonly down-regulated in autism with FMR1-FM and dup(15q). Resultsrepresent means±S.D. of each group. The mean of the value of controlsubjects was set as 1. P-value was calculated by Mann-Whitney U testusing control (N=15) vs. autism with FMR1-FM (N=8) or autism withdup(15q) (N=7). *p<0.05, **p<0.01, ***p<0.001.

FIG. 4 shows that JAKMIP1 and GPR155 were commonly dysregulated byreduction of FMR1 and induction of CYFIP1. SH-SY5Y cells were stablytransfected with i) vector expressing shRNA for control, ii) vectorexpressing shRNA for FMR1, iii) empty expression vector or iv)expression vector for CYFIP1. Total RNA was extracted from each andqRTPCR was performed to validate the effect of FMR1 and CYFIP1 on theexpression of JAKMIP1 and GPR155. A) The expression of FMR1 wassignificantly reduced in SY5Y cells expressing shRNA for FMR1, whereasthe expression of CYFIP1 was significantly induced in SY5Y cellsover-expressing CYFIP1. B) The expression of JAKMIP1 was significantlyreduced in SH-SY5Y cells expressing shRNA for FMR1 and over-expressingCYFIP1. The expression of GPR155 was significantly induced in SH-SY5Ycells expressing shRNA for FMR1 and over-expressing CYFIP1. Resultsrepresent means±S.D. of each group. The mean of the value of eachcontrol was set as 1. Significance was calculated by the Mann-Whitney Utest using SH-SY5Y cells expressing shRNA for control (N=4) vs. shRNAfor FMR1 (N=4) or empty expression vector (N=8) vs. expression vectorfor CYFIP1 (N=7). *P<0.05, **P<0.01.

FIG. 5 shows that the expression of JAKMIP1 protein was dependent onFMR1 and CYFIP1 in mouse cortex and SH-SY5Y cells. Proteins wereextracted from the cortex of FMR1 WT or KO mice (A), or SH-SY5Y cellstransfected with empty vector or CYFIP1 cDNA (B). Western blotting wasperformed to validate the effect of the reduction of FMR1 or inductionof CYFIP1 on the expression of JAKMIP1 protein. The protein expressionof JAKMIP1 was reduced in cortex of FMR1-KO mice (A) as well as SH-SY5Ycells transfected with shRNA for FMR1 and SH-SY5Y over-expressing CYFIP1(B). Data shown in (A) and (B) were the representative of twoindependent experiments.

FIG. 6 shows that JAKMIP1 and GPR155 were dysregulated in the ASDproband in discordant male sib pairs. Total RNA was extracted fromlymphoblastoid cells of 27 male sib pairs discordant for ASD and qRTPCRwere performed to confirm the differential expression of JAKMIP1 andGPR155. Results represent means±S.D. of each group. The mean of thevalue of control subjects was set as 1. P-value was calculated byWilcoxon rank-sum test using control (N=27) vs. ASD (N=27). *p<0.05]

FIG. 7 shows the molecular convergence of FMR1-FM, dup(15q) andidiopathic ASD. The mRNA expression profile in lymphoblastoid cells fromautism with FMR1-FM or dup(15q) and control were compared usingmicroarray analysis. 68 genes were dysregulated in both autism withFMR1-FM and dup(15q). Induction of CYFIP1 in dup(15q) is a potentialmolecular link between FMR1-FM and dup(15q). Among the dysregulatedgenes, JAKMIP1 and GPR155 were further analyzed to confirm the causalrelationship between CYFIP1 and FMR1 expression and their expression inneural cells or tissue and to validate the dysregulation of these genesin lymphoblastoid cells from subjects with idiopathic ASD.

FIG. 8 shows gene networks associated with genes common to FMR1-FM anddup(15q). IPA was used to find significant networks related to the genesdysregulated in both autism with FMR1-FM and dup(15q). Three networkswere identified that contained at least 10 genes. Principal functionsassociated with network A, B and C were cell cycle (P=5.2×10⁻⁸),cellular movement (P=1.3×10⁻⁸) and molecular transport (P=1.6×10⁻⁸), andcell-to-cell signaling and interaction (P=4.3×10⁻⁸), respectively. Genesshown in bold were among the genes commonly dysregulated in autism withFMR1-FM and dup(15q). The intensity of node color indicates the degreeof up-(red) or down-(green) regulation.

FIG. 8 shows differentially expressed probes identified by microarrayanalysis. Venn diagram shows the number of probes differentiallyexpressed in idiopathic ASD (N=15), ASD with FMR1-FM (N=6) and ASD withdup15q (N=7) compared with control (N=15). 124 probes, representing 92genes were identified in common in all three forms of ASD.

FIG. 9 shows a confirmation of the differential gene expression by qPCR.Total RNA was extracted from lymphoblastoid cells from 36 male sib pairsdiscordant for idiopathic ASD. qPCR was performed to confirm thedifferential expression identified by microarray analysis. Bars indicatethe mean of the values of each group. The mean of the value of controlsubjects was set as 0. P-value was calculated by Wilcoxon rank-sum testusing control (N=39) and ASD (N=39). *P<0.05.

FIG. 10 shows gene networks associated with genes dysregulated in commonamong idiopathic ASD, ASD with FMR1-FM and ASD with dup15q. IPA was usedto find significant networks related to the genes dysregulated in commonamong the three different forms of ASD. Four networks were identifiedthat contained at least 10 genes. Principal functions associated withnetwork A, B, C and D were cellular development, cancer, cellulardevelopment and cancer, respectively. Genes shown in bold were among thegenes dysregulated in ASD.

FIG. 11 shows an embodiment of an illustrative computer system that canbe used with embodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art. The techniques and procedures described or referenced hereinare generally well understood and commonly employed using conventionalmethodology by those skilled in the art. As appropriate, proceduresinvolving the use of commercially available kits and reagents aregenerally carried out in accordance with manufacturer defined protocolsand/or parameters unless otherwise noted.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims. It must also be noted that as used herein and in the appendedclaims, the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. All numbersrecited in the specification and associated claims that refer to valuesthat can be numerically characterized with a value other than a wholenumber (e.g. a number of standard deviations from a mean) are understoodto be modified by the term “about”.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. Publications cited herein are citedfor their disclosure prior to the filing date of the presentapplication. Nothing here is to be construed as an admission that theinventors are not entitled to antedate the publications by virtue of anearlier priority date or prior date of invention. Further the actualpublication dates may be different from those shown and requireindependent verification.

Illustrative Embodiments of the Invention

Autism is part of a spectrum of disorders including Asperger syndrome(AS) and other pervasive developmental disorders (PPD). The term“autism” is used herein according to its art accepted meaning andencompasses conditions of impaired social interaction and communicationwith restricted repetitive and stereotyped patterns of behavior,interests and activities present before the age of 3, to the extent thathealth may be impaired. AS is typically distinguished from otherautistic disorders by a lack of a clinically significant delay inlanguage development in the presence of the impaired social interactionand restricted repetitive behaviors, interests, and activities thatcharacterize the autism-spectrum disorder (ASD). PPD-NOS (PPD, nototherwise specified) is typically used to categorize children who do notmeet the strict criteria for autism but who come close, either bymanifesting atypical autism or by nearly meeting the diagnostic criteriain two or three of the key areas.

In about 5 percent of autism cases, another disorder is also present(i.e. an autism-associated disorder). Nearly one-third of those withautism also show signs of epilepsy by adulthood. About 6 percent ofthose with autism also have tuberous sclerosis, a disorder that sharesmany symptoms with autism, including seizures that result from lesions,or cuts on the brain. About 25 percent of persons with autism also havesome degree of mental retardation. About 2 percent of those with autismalso have Fragile X syndrome, the most common inherited form of mentalretardation.

The disclosure provided herein identifies genes that are observed to beDysregulated in Autism Spectrum Disorders. In the instant disclosure,these genes are collectively referred to as “DASD genes” for purposes ofconvenience. Human DASD genes useful in embodiments of the invention areshown for example in Tables 1-6 below as well as the Tables found inNishimura et al., Human Molecular Genetics 2007 16(14): 1682-1698 (thecontents of which are incorporated by reference), disclosure whichincludes information such as the gene name, gene symbol, RefSeq number,and gene locus for these genes. Because the genes disclosed herein areknown in the art and further because of the high level of skillpossessed by artisans in this technical field, the information asdisclosed herein and/or in Nishimura et al., Human Molecular Genetics2007 16(14): 1682-1698 places artisans in possession of thepolynucleotide and polypeptide sequences of these genes by providingthem with the specific disclosure which allows them to retrieve thissequence information from library sources such as GenBank and/orUniProtKB/Swiss-Prot with only minimal effort. As is know in the art,GenBank® is the NIH genetic sequence database, an annotated collectionof all publicly available DNA sequences; and UniProtKB/Swiss-Prot is acurated protein sequence database which provides a high level ofannotation (e.g. technical references describing the features of thesegenes), a minimal level of redundancy and high level of integration withother databases. The DASD gene polynucleotide and polypeptide sequenceinformation can be retrieved from GenBank and/or UniProtKB/Swiss-Protlibrary databases by, for example, querying these databases using theDASD disclosure information as provided herein and/or incorporated byreference into the instant specification (e.g. the gene name, genesymbol, gene RefSeq number, gene locus etc.).

As disclosed in detail below, this disclosure provides methods andmaterials that can be used in the diagnosis and treatment of autismspectrum disorders, and autism-associated disorders. In typicalembodiments of the invention one observes an expression profile of atleast one gene disclosed herein, wherein a dysregulated expressionprofile provides evidence of an autism spectrum disorder. Embodiments ofinvention can be used for example in the diagnosis of (including apredisposition to), and/or treatment of autism spectrum disorders suchas Asperger syndrome, pervasive developmental disorder, mentalretardation, speech delay, and other associated psychiatric andneurological phenomena.

The invention disclosed herein has a number of embodiments. Oneembodiment is a method of identifying a individual having a geneexpression profile associated with autism spectrum disorders comprising:observing an expression profile of at least one DASD gene in a test cell(e.g. mRNA expression in a peripheral blood leukocyte obtained from anindividual suspected of having an autism spectrum); wherein anexpression profile of a DASD gene in the test cell that is at least twostandard deviations from a mean expression of the DASD gene as observedin a control cell (e.g. a peripheral blood leukocyte obtained from anon-effected sibling) identifies the test cell as having a geneexpression profile associated with autism spectrum disorders. In typicalembodiments of the invention, the gene expression profile comprises datarelating to the levels of mRNA expressed by a DASD gene in the cell. Inembodiments of the invention, gene expression can be quantified using acomparison of expression in a test cell relative to a mean expressionobserved in a control cell. For example, in some embodiments of theinvention, the expression of a DASD gene is identified as beingassociated with autism spectrum disorders when it is at least three,four or five standard deviations from the mean expression of the geneobserved in a control cell. In related embodiments of the invention, theexpression of a DASD gene is identified as being associated with autismspectrum disorders when the expression level is at least 20, 30, 40, 50,60 or 70% above or below the expression level of that gene in a controlcell.

Typically in such methods of observing an expression profile of a DASDgene, mRNA expression is observed, for example by using a usingquantitative PCR (qPCR) technique. In certain embodiments of theinvention, the expression profile of the DASD gene in the test cell isobserved using a microarray of polynucleotides. Alternatively, DASDpolypeptide expression is observed, for example by using an antibodyspecific for a polypeptide encoded by a DASD gene (e.g. using an ELISAtechnique or the like). Alternatively, the expression profile isobserved using Southern blotting (e.g. to identify deletions in orduplications of DASD genomic sequences).

Autism spectrum disorder is a heterogeneous condition that appears toresult from the combined effects of multiple, subtle genetic changesinteracting with environmental factors. Consequently, in someembodiments of the invention, an expression profile of at least, 2, 3,4, 5, 6, 7, 8, 9 or 10 or more DASD genes are observed in order toobtain a detailed profile of these multiple genetic changes and/or tostratify individuals into subsets of autism spectrum disorders (e.g.using microarray technologies). For example, in certain embodiments ofthe invention, the method is performed on a plurality of individuals andthen segregated based upon similarities or differences in their geneexpression profiles. Optionally, the expression profile(s) of the testmammalian cell is observed using a computer system comprising aprocessor element and a memory storage element adapted to process andstore data from one or more expression profiles (e.g. in a library ofsuch profiles). In this context, one embodiment of the inventioncomprises an electronically searchable library of profiles, wherein theprofiles include individual's gene expression data in combination withother diagnostic data, for example assessments of whether the individualexhibits behavior associated with an autism spectrum disorder (e.g.behavioral test data such as that obtained in an Autism DiagnosticInterview (ADI-R)).

In typical embodiments of the invention, these methods are used tofacilitate diagnosis of an autism spectrum disorder in an individual. Inthis context, a cell examined in the methods of the invention can be aleukocyte obtained from the peripheral blood of the individual. Incertain embodiments of the invention, the test cell is obtained from anindividual previously identified as exhibiting a behavior associatedwith autism spectrum disorders. In some embodiments of the invention,the test cell is obtained from an individual identified as having afamily member previously identified as exhibiting a behavior associatedwith autism spectrum disorders. In typical embodiments, the controlmammalian cell is obtained from an individual previously identified asnot exhibiting a behavior associated with autism spectrum disorders.Embodiments of the invention include methods which perform a furtherdiagnostic procedure for autism spectrum disorders on an individualidentified as having a gene expression profile associated with autismspectrum disorders (e.g. a procedure following standard validatingmeasures, such as the Autism Diagnostic Interview (ADI-R)). Optionally,the test mammalian cell and the control mammalian cell are obtained fromindividuals who are related as siblings or as a parent and a child.

Embodiments of the invention further include a kit comprising: a firstcontainer, a label on said container, and a composition contained withinsaid container; wherein the composition includes polymerase chainreaction (PCR) primer effective in the quantitative real time analysisof the mRNA expression levels of one or more DASD genes, the label onsaid container, or a package insert included in said container indicatesthat the composition can be used to observe expression levels of one ormore DASD genes in at least one type of human leukocyte; a secondcontainer comprising a pharmaceutically-acceptable buffer; andinstructions for using the PCR primer to obtain an expression profile ofthe one or more DASD genes. Optionally the kit comprises 2, 3, 4, 5, 6,7, 8, 9 or 10 polymerase chain reaction (PCR) primers effective in thequantitative real time analysis of the mRNA expression levels ofdifferent DASD genes.

In certain embodiments of the invention, a kit further comprises acomputer readable a memory storage element adapted to process and storedata from one or more expression profiles. In some of these embodiments,the memory storage element organizes expression profile data into aformat adapted for electronic comparisons with a library of expressionprofile data.

Embodiments of the invention further comprise, for example, methods ofassessing the response of a subject to a treatment of an autism spectrumdisorder, or an autism-associated disorder (e.g. treatment comprisingthe administration of a therapeutic agent), the method comprisingdetecting altered DASD gene or polypeptide expression in a sample fromthe treated subject, the presence of the alteration being indicative ofa response to the treatment.

As noted above, embodiments of the invention compare DASD geneexpression in a test cell (e.g. a cell obtained from an individualsuspected of having an autism spectrum disorder) with DASD geneexpression in a normal cell (e.g. a cell obtained from an individual nothaving an autism spectrum disorder) in order to determine if the testcell exhibits altered DASD gene expression. In addition to using normalcells as a comparative sample for DASD expression, in certain situationsone can also use a predetermined normative value such as a predeterminednormal sequence, and/or level of DASD mRNA or polypeptide expression(see, e.g., Grever et al., J. Comp. Neurol. 1996 Dec. 9; 376(2):306-14and U.S. Pat. No. 5,837,501) to evaluate levels of DASD expression in agiven sample. The term “status” in this context is used according to itsart accepted meaning and refers to the condition or state of a gene andits products. Typically, skilled artisans use a number of parameters toevaluate the condition or state of a gene and its products. Theseinclude, but are not limited to the level, sequence of and biologicalactivity of expressed gene products (such as DASD mRNA, polynucleotidesand polypeptides). In certain embodiments of the invention, theexpression of a DASD gene product is characterized by observing how farthe expression level of a DASD mRNA in a sample deviates from a meanexpression level of that mRNA in control cells in order to obtain astatistical measure of precision. Standard deviation is a measure of thevariability or dispersion of a data set, in this case, the levels ofmRNA expression of selected genes. Standard deviation in this contextallows determinations of how spread out a set of expression values isand how a given sample fits into such analyses. Illustrative statisticalmethods for determining such values can be found for example in Cui etal., Genome Biol. (2003) 4:210; Tusher et al., Proc. Natl Acad. Sci. USA(2001) 98:5116-5121; Jeffery et al., BMC Bioinformatics (2006) 7:359;and Breitling et al., FEBS Lett. (2004) 573:83-92, the contents of whichare incorporated by reference.

As discussed in detail below, the status of a DASD gene can be analyzedby a number of techniques that are well known in the art. Typicalprotocols for evaluating the status of the DASD gene and gene productsare found, for example in Ausubel et al. eds., 1995, Current ProtocolsIn Molecular Biology, Units 2 (Northern Blotting), 4 (SouthernBlotting), 15 (Immunoblotting) and 18 (PCR Analysis). The status of aDASD gene in a biological sample is evaluated by various methodsutilized by skilled artisans including, but not limited to genomicSouthern analysis (to examine, for example perturbations in DASD genomicsequences), Northern analysis and/or PCR analysis of DASD mRNA (toexamine, for example alterations in the polynucleotide sequences orexpression levels of DASD mRNAs), and, Western and/orimmunohistochemical analysis (to examine, for example alterations inpolypeptide sequences, alterations in expression levels of DASD proteinsetc.). Detectable DASD polynucleotides include, for example, a DASD geneor fragment thereof, DASD mRNA, alternative splice variants, DASD mRNAs,and recombinant DNA or RNA molecules comprising a DASD polynucleotide.

By examining a biological sample obtained from an individual (e.g. aperipheral blood leukocyte) for evidence altered gene expression of oneor more genes whose expression is dysregulated in individuals diagnosedwith autism spectrum disorders, medical personnel can obtain informationuseful in the identification, treatment and/or management of thesedisorders. Typically, the methods comprise detecting in a sample from asubject the presence of altered DASD gene expression, the presence ofthe alteration being indicative of the presence of, or predisposition toautism, an autism spectrum disorder, or an autism-associated disorder.In this context, “altered gene expression” encompasses altered DASD mRNAand/or polypeptide levels; altered DASD polynucleotide and polypeptidesequences, altered DASD genomic DNA methylation patterns and the like,alterations that are typically absent in individuals not having anautism spectrum disorder. In such examinations, the status of one ormore DASD polynucleotides and/or polypeptides in a biological sample ofinterest (e.g. a peripheral blood leukocyte obtained from an individualsuspected of having an autism spectrum disorder) can be compared to astandard or control, for example, or to the status of the DASDpolynucleotide(s) or polypeptide(s) in a corresponding normal sample(e.g. a peripheral blood leukocyte obtained from a non-effected siblingor another individual not having a autism spectrum disorder). Analteration in the status of DASD gene expression in the biologicalsample (as compared to a control or standardized sample and/or value)then provides evidence of an autism spectrum disorder.

As noted above, embodiments of invention provide methods that comprisefor example observing the expression status of one or more DASD genes ina subject in order to obtain diagnostically and/or prognostically usefulinformation. Such methods typically use a leukocyte obtained from asubject to assess the status of a DASD gene. The sample may be anybiological sample derived from a subject, which contains nucleic acidsor polypeptides. Examples of such samples include fluids, tissues, cellsamples, organs, biopsies, etc. Most preferred samples are blood andother leukocyte containing tissues etc. Pre-natal diagnosis may also beperformed by testing for example fetal cells or placental cells. Anybiological sample from which DASD genes and/or the products of DASDgenes can be isolated is suitable. The sample may be collected accordingto conventional techniques and used directly for diagnosis or stored.The sample may be treated prior to performing the method, in order torender or improve availability of nucleic acids and/or polypeptides fortesting. Treatments include, for example, lysis (e.g., mechanical,physical, chemical, etc.), centrifugation, etc. Also, the nucleic acidsand/or polypeptides may be pre-purified or enriched by conventionaltechniques, and/or reduced in complexity. Nucleic acids and polypeptidesmay also be treated with enzymes or other chemical or physicaltreatments to produce fragments thereof.

The isolation of biological samples from a subject which contain nucleicacids and/or polypeptides is well know in the art. For example, certainembodiments isolate leukocytes from the circulating blood in order toassess the status of DASD genes in these cells. In such embodiments,blood is typically collected from subjects into heparinized bloodcollection tubes by personnel trained in phlebotomy using steriletechnique. The collected blood samples can be divided into aliquots andcentrifuged, and the buffy coat layer can then be removed (this fractioncontains the leukocytes). RNA can then be extracted using a commercialRNA purification kit (e.g. RNeasy; Qiagen, Valencia, Calif.). RNAquality can be determined, for example, with an A260/A280 ratio andcapillary electrophoresis on an apparatus such as an Agilent 2100Bioanalyzer automated analysis system (Agilent Technologies, Palo Alto,Calif.).

In typical embodiments of the invention, a sample is contacted withreagents such as probes, primers or ligands (e.g. antibodies) in orderto assess the presence of altered gene expression of a DASD gene. Suchmethods may be performed by a wide variety of apparatuses used in theart, such as a plate, tube, well, glass, etc. In specific embodiments,the contacting is performed on a substrate coated with the reagent, suchas a nucleic acid array or a specific ligand (e.g. antibody) array. Thesubstrate may be solid or semi-solid substrate such as any supportcomprising glass, plastic, nylon, paper, metal, polymers and the like.The substrate may be of various forms and sizes, such as a chip, aslide, a membrane, a bead, a column, a gel, etc. The contacting may bemade under any condition suitable for a complex to be formed between thereagent and the nucleic acids or polypeptides of the sample.

A wide variety of methods known in the art can be used to examine theexpression of DASD polypeptides and polynucleotides in cells such asperipheral blood leukocytes. For example, certain embodiments of methodswhich examine DASD polynucleotides and polypeptides in such cells areanalogous to those methods from well-established diagnostic assays knownin the art such as those that observe the expression of biomarkers suchas prostate specific antigen (PSA) polynucleotides and polypeptides. Forexample, just as PSA polynucleotides are used as probes (for example inNorthern analysis, see, e.g., Sharief et al., Biochem. Mol. Biol. Int.33(3):567-74(1994)) and primers (for example in PCR analysis, see, e.g.,Okegawa et al., J. Urol. 163(4): 1189-1190 (2000)) to observe thepresence and/or the level of PSA mRNAs in methods of monitoring PSAexpression, the DASD polynucleotides identified herein can be utilizedin the same way to observe DASD overexpression or underexpression orother alterations in these genes. Similarly, just as PSA polypeptidesare used to generate antibodies specific for PSA which can then be usedto observe the presence and/or the level of PSA proteins in methods tomonitor PSA protein expression (see, e.g., Stephan et al., Urology55(4):560-3 (2000)) in prostate cells (see, e.g., Alanen et al., Pathol.Res. Pract. 192(3):233-7 (1996)), the DASD polypeptides described hereincan be utilized to generate antibodies for use in detecting DASDexpression in peripheral blood leukocytes and the like. Accordingly, thestatus of DASD gene products provides information useful for predictinga variety of factors including the presence of and/or susceptibility toautism spectrum disorders. As discussed in detail herein, the status ofDASD gene products in patient samples can be analyzed by a varietyprotocols that are well known in the art including immunohistochemicalanalysis, the variety of Northern blotting techniques including in situhybridization, RT-PCR analysis (e.g. quantitative RT-PCR), Western blotanalysis, polynucleotide and polypeptide microarray analysis and thelike.

Exemplary embodiments of the invention include methods for identifying acell that overexpresses or underexpresses DASD polynucleotides and/orpolypeptides. One such embodiment of the invention is an assay thatquantifies the expression of the DASD gene in a cell by detecting theabsence/presence and/or relative levels of DASD mRNA concentrations inthe cell. Methods for the evaluation of particular mRNAs in cells arewell known and include, for example, hybridization assays usingcomplementary DNA probes (such as in situ hybridization using labeledDASD riboprobes, Northern blot and related techniques) and variousnucleic acid amplification assays (such as qPCR using complementaryprimers specific for DASD, and other amplification type detectionmethods, such as, for example, branched DNA, SISBA, TMA and the like).

Embodiments of the invention include methods for detecting a DASD mRNAin a biological sample by generating cDNA in the sample by reversetranscription using at least one primer; amplifying the cDNA so producedusing an DASD polynucleotides as sense and antisense primers to amplifyDASD cDNAs therein; and detecting the presence of the amplified DASDcDNA. One exemplary PCR method that can be used in embodiments of theinvention is a real-time quantitative PCR (qPCR) assay. Such real-timeassays provide a large dynamic range of detection and a highly sensitivemethods for determining the amount of DNA template of interest. WhenqPCR follows a reverse transcription reaction, it can be used toquantify RNA templates as well. In addition, qPCR makes quantificationof DNA and RNA much more precise and reproducible because it relies onthe analysis of PCR kinetics rather than endpoint measurements.Illustrative qPCR assays are disclosed for example in U.S. PatentApplication Nos.: 2006/0008809; 2003/0219788; 2006/0051787; and2006/0099620, the contents of which are incorporated by reference.

Some embodiments of the invention can use next-generation sequencingtechnologies for the expression profiling of DASD genes, for examplethose that are commercially available from vendors such as APPLIEDBIOSYSTEMS and ILLUMINA. Typically in these embodiments, one can countthe number of copies of each DASD gene that is expressed in order toprovide assays that quantify the expression levels of all mRNA moleculesin a cell. Because such methods are based on sequencing and nothybridization, they can provide an unbiased, probe-less measurement ofall mRNA molecules in a sample. Illustrative aspects of suchtechnologies are disclosed for example in U.S. Patent No. 20080262747,the contents of which are incorporated by reference.

Another embodiment of the invention is a method of detecting DASD geneshaving altered copy numbers (i.e. genes having a copy numbers that isabove or below the number of copies observed in cells obtained fromnormal individuals) and/or another chromosomal rearrangement in abiological sample by isolating genomic DNA from the sample; amplifyingthe isolated genomic DNA using DASD polynucleotides as sense andantisense primers; and detecting the presence of the altered DASD gene.Any number of appropriate sense and antisense probe combinations can bedesigned from the nucleotide sequence provided for the DASD and used forthis purpose.

The invention also provides assays for detecting the presence of a DASDprotein in a tissue or other biological sample and the like. Methods fordetecting a DASD-related protein are also well known and include, forexample, immunoprecipitation, immunohistochemical analysis, Western blotanalysis, molecular binding assays, ELISA, ELIFA and the like. Forexample, a method of detecting the presence of a DASD-related protein ina biological sample comprises first contacting the sample with a DASDantibody, a DASD-reactive fragment thereof, or a recombinant proteincontaining an antigen binding region of a DASD antibody; and thendetecting the binding of DASD-related protein in the sample. Optionally,DASD polypeptide expression is measured in a tissue microarray.

In another embodiment of the invention, one can evaluate the status DASDnucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules. Theseperturbations can include insertions, deletions, substitutions,duplications and the like in the coding and regulatory regions of theDASD gene. Such evaluations are useful because perturbations in thenucleotide and amino acid sequences are observed in a large number ofproteins associated with a growth dysregulated phenotype (see, e.g.,Marrogi et al., 1999, J. Cutan. Pathol. 26(8):369-378). For example, amutation in the sequence of an DASD 5′ or 3′ regulatory enhancer and/orpromoter sequence may provide evidence of dysregulated expression. Suchassays therefore have diagnostic and predictive value where a mutationin DASD is indicative of dysregulated expression.

A wide variety of assays for observing perturbations in nucleotide andamino acid sequences are well known in the art. For example, the sizeand structure of nucleic acid or amino acid sequences of DASD geneproducts are observed by the Northern, Southern, Western, PCR and DNAsequencing protocols discussed herein. In addition, other methods forobserving perturbations in nucleotide and amino acid sequences such assingle strand conformation polymorphism analysis are well known in theart (see, e.g., U.S. Pat. Nos. 5,382,510 and 5,952,170, the contents ofwhich are incorporated by reference).

The mutation in a DASD gene may be a single base substitution mutationresulting in an amino acid substitution, a single base substitutionmutation resulting in a translational stop, an insertion mutation, adeletion mutation, or a gene rearrangement. The mutation may be locatedin an intron, an exon of the gene, or a promotor or other regulatoryregion which affects the expression of the gene. Screening for mutatednucleic acids can be accomplished by direct sequencing of nucleic acids.Nucleic acid sequences can be determined through a number of differenttechniques which are well known to those skilled in the art, for exampleby chemical or enzymatic methods. The enzymatic methods rely on theability of DNA polymerase to extend a primer, hybridized to the templateto be sequenced, until a chain-terminating nucleotide is incorporated.The most common methods utilize dideoxynucleotides. Primers may belabelled with radioactive or fluorescent labels. Various DNA polymerasesare available including Klenow fragment, AMV reverse transcriptase,Thermus aquaticus DNA polymerase, and modified T7 polymerase.

Ligase chain reaction (LCR) is yet another method of screening formutated nucleic acids. LCR can be carried out in accordance with knowntechniques and is especially useful to amplify, and thereby detect,single nucleotide differences between two DNA samples. In general, thereaction is carried out with two pairs of oligonucleotide probes: onepair binds to one strand of the sequence to be detected; the other pairbinds to the other strand of the sequence to be detected. The reactionis carried out by, first, denaturing (e.g., separating) the strands ofthe sequence to be detected, then reacting the strands with the twopairs of oligonucleotide probes in the presence of a heat stable ligaseso that each pair of oligonucleotide probes hybridize to target DNA and,if there is perfect complementarity at their junction, adjacent probesare ligated together. The hybridized molecules are then separated underdenaturation conditions. The process is cyclically repeated until thesequence has been amplified to the desired degree. Detection may then becarried out in a manner like that described above with respect to PCR.

Southern hybridization is also an effective method of identifyingdifferences in sequences. Hybridization conditions, such as saltconcentration and temperature can be adjusted for the sequence to bescreened. Southern blotting and hybridizations protocols are describedin Current Protocols in Molecular Biology (Greene Publishing Associatesand Wiley-Interscience), pages 2.9.1-2.9.10. Probes can be labelled forhybridization with random oligomers (primarily 9-mers) and the Klenowfragment of DNA polymerase. Very high specific activity probe can beobtained using commercially available kits such as the Ready-To-Go DNALabelling Beads (Pharmacia Biotech), following the manufacturer'sprotocol. Briefly, 25 ng of DNA (probe) is labelled with ³²P-dCTP in a15 minute incubation at 37° C. Labelled probe is then purified over aChromaSpin (Clontech) nucleic acid purification column.

Determinations of the presence of the polymorphic form of a DASD proteincan also be carried out, for example, by isoelectric focusing, proteinsizing, or immunoassay. In an immunoassay, an antibody that selectivelybinds to the mutated protein can be utilized (for example, an antibodythat selectively binds to the mutated form of DASD encoded protein).Such methods for isoelectric focusing and immunoassay are well known inthe art. For example, changes resulting in amino acid substitutions,where the substituted amino acid has a different charge than theoriginal amino acid, can be detected by isoelectric focusing.Isoelectric focusing of the polypeptide through a gel having anampholine gradient at high voltages separates proteins by their pI. ThepH gradient gel can be compared to a simultaneously run gel containingthe wild-type protein. Protein sizing techniques such as proteinelectrophoresis and sizing chromatography can also be used to detectchanges in the size of the product.

As an alternative to isoelectric focusing or protein sizing, the step ofdetermining the presence of the mutated polypeptides in a sample may becarried out by an antibody assay with an antibody which selectivelybinds to the mutated polypeptides (i.e., an antibody which binds to themutated polypeptides but exhibits essentially no binding to thewild-type polypeptide without the polymorphism in the same bindingconditions). Antibodies used to selectively bind the products of themutated genes can be produced by any suitable technique. For example,monoclonal antibodies may be produced in a hybridoma cell line accordingto the techniques of Kohler and Milstein, Nature, 265, 495 (1975), whichis hereby incorporated by reference. A hybridoma is an immortalized cellline which is capable of secreting a specific monoclonal antibody. Themutated products of genes which are associated with autism may beobtained from a human patient, purified, and used as the immunogen forthe production of monoclonal or polyclonal antibodies. Purifiedpolypeptides may be produced by recombinant means to express abiologically active isoform, or even an immunogenic fragment thereof maybe used as an immunogen. Monoclonal Fab fragments may be produced inEscherichia coli from the known sequences by recombinant techniquesknown to those skilled in the art.

Additionally, one can examine the methylation status of the DASD gene ina biological sample. Aberrant demethylation and/or hypermethylation ofCpG islands in gene 5′ regulatory regions frequently occurs inimmortalized and transformed cells, and can result in altered expressionof various genes. For example, promoter hypermethylation of the pi-classglutathione S-transferase (a protein expressed in normal prostate butnot expressed in >90% of prostate carcinomas) appears to permanentlysilence transcription of this gene and is the most frequently detectedgenomic alteration in prostate carcinomas (De Marzo et al., Am. J.Pathol. 155(6): 1985-1992 (1999)). A variety of assays for examiningmethylation status of a gene are well known in the art. For example, onecan utilize, in Southern hybridization approaches, methylation-sensitiverestriction enzymes which cannot cleave sequences that containmethylated CpG sites to assess the methylation status of CpG islands. Inaddition, MSP (methylation specific PCR) can rapidly profile themethylation status of all the CpG sites present in a CpG island of agiven gene. This procedure involves initial modification of DNA bysodium bisulfite (which will convert all unmethylated cytosines touracil) followed by amplification using primers specific for methylatedversus unmethylated DNA. Protocols involving methylation interferencecan also be found for example in Current Protocols In Molecular Biology,Unit 12, Frederick M. Ausubel et al. eds., 1995.

Embodiments of the invention include compositions that can be used forexample in various methods disclosed herein. Compositions useful in themethods disclosed herein typically include for example one or more DASDnucleic acid molecules designed for use as a probe such as a PCR primerin a method used to monitor DASD mRNAs or genomic sequences in a cell.Optionally, the probe or primer has 8, 9, 19, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides that arecomplementary to a DASD mRNA. In certain embodiments, the probe orprimer comprises 5-25 heterologous polynucleotide sequences (e.g. tofacilitate cloning). Typically, the probe or primer will hybridize tothe DASD mRNA under “stringent conditions” i.e. those that: (1) employlow ionic strength and high temperature for washing, for example 0.015 Msodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at50° C.; (2) employ during hybridization a denaturing agent, such asformamide, for example, 50% (v/v) formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS,and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC(sodium chloride/sodium. citrate) and 50% formamide at 55° C., followedby a high-stringency wash consisting of 0.1×SSC containing EDTA at 55°C.

Specifically contemplated nucleic acid related embodiments of theinvention disclosed herein are genomic DNA, cDNAs, ribozymes, andantisense molecules, as well as nucleic acid molecules based on analternative backbone, or including alternative bases, whether derivedfrom natural sources or synthesized, and include molecules capable ofinhibiting the RNA or protein expression of DASD. For example, antisensemolecules can be RNAs or other molecules, including peptide nucleicacids (PNAs) or non-nucleic acid molecules such as phosphorothioatederivatives, that specifically bind DNA or RNA in a base pair-dependentmanner. Compositions of the invention include one or more antibodiesthat bind DASD and which can be used as a probe to monitor DASDpolypeptide expression in a cell. A skilled artisan can readily preparethese polynucleotide and polypeptide compounds using the DASDpolynucleotides and polynucleotide sequences and associated informationthat is disclosed herein.

For use in the methods described above, kits are also provided by theinvention. Such kits may comprise a carrier means beingcompartmentalized to receive in close confinement one or more containermeans such as vials, tubes, and the like, each of the container meanscomprising one of the separate elements to be used in the method. Forexample, one of the container means may comprise a probe that is or canbe detectably labeled. Such probe may be an antibody or polynucleotidespecific for DASD protein or DASD gene or message, respectively. Wherethe kit utilizes nucleic acid hybridization to detect the target nucleicacid, the kit may also have containers containing nucleotide(s) foramplification of the target nucleic acid sequence and/or a containercomprising a reporter-means, such as a biotin-binding protein, such asavidin or streptavidin, bound to a reporter molecule, such as anenzymatic, florescent, or radioisotope label.

The kits of the invention have a number of embodiments. A typicalembodiment is a kit comprising a container, a label on the container,and a composition contained within the container; wherein thecomposition includes: (1) a polynucleotide that hybridizes to acomplement of the DASD polynucleotide and/or (2) an antibody that bindsthe DASD polypeptide, the label on the container indicates that thecomposition can be used to evaluate the expression level of the DASDgene product in at least one type of mammalian cell (e.g. a humanperipheral blood leukocyte), and instructions for using the DASDpolynucleotide or antibody for evaluating the presence of DASD RNA, DNAor protein in at least one type of mammalian cell.

Autism is a heterogeneous condition and is likely to result from thecombined effects of multiple, genetic changes including copy numbervariations and single nucleotide polymorphisms, interacting withenvironmental factors (see, e.g. Folstein et al., (2001) Nat. Rev.Genet., 2, 943-955; Belmonte et al., (2004) Mol. Psychiatry, 9, 646-663;Veenstra-Vanderweele et al., (2004) Annu Rev Genomics Hum Genet, 5,379-405; and Muhle et al., (2004) Pediatrics, 113, e472-486).Classifications such as a computer based hierarchy of autistic patientsbased on genotypic and phenotypic information is one effective way toidentify more homogeneous subgroups and hasten the identification ofgenes underlying autism (see, e.g. Folstein et al., (2001) Nat. Rev.Genet., 2, 943-955; Belmonte et al., (2004) Mol. Psychiatry, 9, 646-663;Veenstra-Vanderweele et al., (2004) Annu Rev Genomics Hum Genet, 5,379-405; and Muhle et al., (2004) Pediatrics, 113, e472-486). About 3%of autistic children have either FMR1-FM or dup(15q), thus comprisingmore homogeneous populations with a single major genetic etiology fortheir autism.

In this context, embodiments of the invention further provides methodsof obtaining a gene expression profile associated with autism spectrumdisorders and methods of generating a database, or collection, of suchprofiles. The methods generally involve observing a gene expressionprofile associated with autism spectrum disorders, storing the data on acomputer readable medium (CRM), and linking the data with at least oneadditional data point such as an individual identifying code and/orfamilial genetic information and/or the presence or absence of otherphenomena (e.g. behavioral phenomena) associated with autism spectrumdisorders such as Asperger syndrome, pervasive developmental disorder,mental retardation, speech delay, and other associated psychiatric andneurological phenomena. The profile having this information is thenrecorded on a CRM.

Computer related embodiments of the invention disclosed herein can beperformed for example, using one of the many computer systems known inthe art. For example, embodiments of the invention can include asearchable database library comprising a plurality of cell profilesrecorded on a computer readable medium, each of the profiles comprisingfurther information such as identifying codes and/or familialrelationships and/or gene expression and/or behavioral phenomenaassociated with autism spectrum disorders. In this context, one can thenuse this library of gene expression and behavioral data to, for example,classify and/or examine etiological subsets of autism as well as toexplore the pathophysiology of this condition. In one embodiment of theinvention, data obtained from a new test sample is compared to data insuch a library in order to, for example, find similar comparativeprofiles in the library from which diagnostic and/or prognosticinformation can be inferred. FIG. 11 illustrates an exemplarygeneralized computer system 202 that can be used to implement elementsthe present invention. The computer 202 typically comprises a generalpurpose hardware processor 204A and/or a special purpose hardwareprocessor 204B (hereinafter alternatively collectively referred to asprocessor 204) and a memory 206, such as random access memory (RAM). Thecomputer 202 may be coupled to other devices, including input/output(I/O) devices such as a keyboard 214, a mouse device 216 and a printer228.

In one embodiment, the computer 202 operates by the general purposeprocessor 204A performing instructions defined by the computer program210 under control of an operating system 208. The computer program 210and/or the operating system 208 may be stored in the memory 206 and mayinterface with the user and/or other devices to accept input andcommands and, based on such input and commands and the instructionsdefined by the computer program 210 and operating system 208 to provideoutput and results. Output/results may be presented on the display 222or provided to another device for presentation or further processing oraction. In one embodiment, the display 222 comprises a liquid crystaldisplay (LCD) having a plurality of separately addressable liquidcrystals. Each liquid crystal of the display 222 changes to an opaque ortranslucent state to form a part of the image on the display in responseto the data or information generated by the processor 204 from theapplication of the instructions of the computer program 210 and/oroperating system 208 to the input and commands. The image may beprovided through a graphical user interface (GUI) module 218A. Althoughthe GUI module 218A is depicted as a separate module, the instructionsperforming the GUI functions can be resident or distributed in theoperating system 208, the computer program 210, or implemented withspecial purpose memory and processors.

Some or all of the operations performed by the computer 202 according tothe computer program 210 instructions may be implemented in a specialpurpose processor 204B. In this embodiment, some or all of the computerprogram 210 instructions may be implemented via firmware instructionsstored in a read only memory (ROM), a programmable read only memory(PROM) or flash memory in within the special purpose processor 204B orin memory 206. The special purpose processor 204B may also be hardwiredthrough circuit design to perform some or all of the operations toimplement the present invention. Further, the special purpose processor204B may be a hybrid processor, which includes dedicated circuitry forperforming a subset of functions, and other circuits for performing moregeneral functions such as responding to computer program instructions.In one embodiment, the special purpose processor is an applicationspecific integrated circuit (ASIC).

The computer 202 may also implement a compiler 212 which allows anapplication program 210 written in a programming language such as COBOL,C++, FORTRAN, or other language to be translated into processor 204readable code. After completion, the application or computer program 210accesses and manipulates data accepted from I/O devices and stored inthe memory 206 of the computer 202 using the relationships and logicthat was generated using the compiler 212. The computer 202 alsooptionally comprises an external communication device such as a modem,satellite link, Ethernet card, or other device for accepting input fromand providing output to other computers.

In one embodiment, instructions implementing the operating system 208,the computer program 210, and the compiler 212 are tangibly embodied ina computer-readable medium, e.g., data storage device 220, which couldinclude one or more fixed or removable data storage devices, such as azip drive, floppy disc drive 224, hard drive, CD-ROM drive, tape drive,etc. Further, the operating system 208 and the computer program 210 arecomprised of computer program instructions which, when accessed, readand executed by the computer 202, causes the computer 202 to perform thesteps necessary to implement and/or use the present invention or to loadthe program of instructions into a memory, thus creating a specialpurpose data structure causing the computer to operate as a speciallyprogrammed computer executing the method steps described herein.Computer program 210 and/or operating instructions may also be tangiblyembodied in memory 206 and/or data communications devices 230, therebymaking a computer program product or article of manufacture according tothe invention. As such, the terms “article of manufacture,” “programstorage device” and “computer program product” as used herein areintended to encompass a computer program accessible from any computerreadable device or media.

Of course, those skilled in the art will recognize that any combinationof the above components, or any number of different components,peripherals, and other devices, may be used with the computer 202.Although the term “user computer” is referred to herein, it isunderstood that a user computer 102 may include portable devices such asmedication infusion pumps, analyte sensing apparatuses, cellphones,notebook computers, pocket computers, or any other device with suitableprocessing, communication, and input/output capability.

Yet another embodiment of this invention comprises a method of screeningfor a compound that modulates DASD protein expression comprising thesteps of contacting a cell that expresses an endogenous or exogenousDASD protein with one or more compounds and then determining if the oneor more compounds modulates DASD protein expression in the cell (e.g. byqPCR techniques practiced on the cell in the presence and absence of theone or more compounds). Another embodiment of this invention comprises amethod of screening for a compound that interacts with an DASD proteincomprising the steps of contacting one or more compounds with the DASDprotein, and then determining if a compound interacts with the DASDprotein (e.g. by binding techniques that separating compounds thatinteract with the DASD protein from compounds that do not). Thisembodiment of the invention can be used for example to screen chemicallibraries for compounds which modulate, e.g., inhibit, antagonize, oragonize or mimic, the expression of a DASD as measured by one of theassays disclosed herein. The chemical libraries can be peptidelibraries, peptidomimetic libraries, chemically synthesized libraries,recombinant, e.g., phage display libraries, and in vitrotranslation-based libraries, other non-peptide synthetic organiclibraries. Exemplary libraries are commercially available from severalsources (e.g. e, Tripos/PanLabs, ChemDesign, Pharmacopoeia). Typicalpeptide libraries and screening methods that can be used to identifycompounds that modulate the expression of and/or interact with DASDprotein sequences are disclosed for example in U.S. Pat. Nos. 5,723,286and 5,733,731, the contents of which are incorporated by reference.

Various aspects of the invention are further described and illustratedby way of the examples that follow, none of which are intended to limitthe scope of the invention. Certain disclosure in the examples below canbe found in Nishimura et al., Human Molecular Genetics 2007 16(14):1682-1698, the contents of which are incorporated by reference. Inaddition, certain methods and materials used in embodiments of theinvention can be those found for example n U.S. Patent Application Nos.:2002/0155450; 2006/0141519; 2007/0134664; and 2009/0011414, the contentsof which are incorporated by reference.

EXAMPLES Example 1 Genome-Wide Expression Profiling of LymphoblastoidCell Lines Distinguishes Different Forms of Autism and Reveals SharedPathways

Autism is a heterogeneous condition that is likely to result from thecombined effects of multiple genetic factors interacting withenvironmental factors. Given its complexity, the study of autismassociated with Mendelian single gene disorders or known chromosomaletiologies provides an important perspective. We used microarrayanalysis to compare the mRNA expression profile in lymphoblastoid cellsfrom males with autism due to a Fragile X mutation (FMR1-FM), or a15q11-q13 duplication (dup(15q)), and non-autistic controls. We wereable to clearly distinguish autism from controls and separateindividuals with autism based on their genetic etiology. Sixty-eightgenes were dysregulated in common between autism with FMR1-FM anddup(15q). We identified a potential molecular link between FMR1-FM anddup(15q), the cytoplasmic FMR1 interacting protein 1 (CYFIP1), which wasup-regulated in dup(15q) patients. We were able to confirm this link invitro by showing common regulation of two other dysregulated genes,JAKMIP1 and GPR155, downstream of FMR1 and CYFIP1. We also confirmed thereduction of the JAKMIP1 protein in FMR1 knock out mice, demonstratingin vivo relevance. Finally, we showed independent confirmation of rolesfor JAKMIP1 and GPR155 in autism spectrum disorders (ASD) by showingtheir differential expression in male sib pairs discordant foridiopathic ASD. These results provide evidence that blood-derivedlymphoblastoid cells gene expression is likely to be useful foridentifying etiological subsets of autism and to explore itspathophysiology.

It has become increasingly clear that genetic factors are significantdeterminants of autism pathophysiology (see, e.g. Folstein et al.,(2001) Nat. Rev. Genet., 2, 943-955; Belmonte et al., (2004) Mol.Psychiatry, 9, 646-663; Veenstra-Vanderweele et al., (2004) Annu RevGenomics Hum Genet, 5, 379-405; Muhle et al., (2004) Pediatrics, 113,e472-486). Although, multiple genetic approaches have been undertaken toidentify loci or genes for autism spectrum disorders (ASD) (1-18),identification of causal genes has been hampered by genetic andphenotypic heterogeneity. Thus, it seems reasonable to accelerate thegene discovery process by using combinations of experimental approaches,such as the study of “single gene” or more simple causes, such aschromosomal copy number imbalances, whose phenotypes include ASD (see,e.g. Folstein et al., (2001) Nat. Rev. Genet., 2, 943-955; Belmonte etal., (2004) Mol. Psychiatry, 9, 646-663; Veenstra-Vanderweele et al.,(2004) Annu Rev Genomics Hum Genet, 5, 379-405; Muhle et al., (2004)Pediatrics, 113, e472-486). One such disorder is fragile X syndrome(FXS) (see, e.g. Folstein et al., (2001) Nat. Rev. Genet., 2, 943-955;Belmonte et al., (2004) Mol. Psychiatry, 9, 646-663;Veenstra-Vanderweele et al., (2004) Annu Rev Genomics Hum Genet, 5,379-405; Muhle et al., (2004) Pediatrics, 113, e472-486; and Brown etal. (1986) Am. J. Med. Genet., 23, 341-352), which is caused by anexpansion of the trinucleotide repetitive sequence (CGG)n in thepromoter region of the fragile X mental retardation 1 (FMR1) genelocated at Xq27.3 (see. e.g. Verkerk et al. (1991) Cell, 65, 905-914).This mutation causes a significant deficit of the FMR1 protein (FMRP)and a phenotype including cognitive impairment and other behavioralabnormalities that overlap with ASD. The prevalence of ASD among FXScases has been estimated at 15-33% (see, e.g. Rogers et al. (2001) J.Dev. Behav. Pediatr., 22, 409-417; and Goodlin-Jones et al. (2004) J.Dev. Behav. Pediatr., 25, 392-398) and approximately 1% to 2% of thosewith autism and no obvious physical features of FXS are found to haveFMR1-FM (see, e.g. Brown et al. (1986) Am. J. Med. Genet., 23, 341-352;and Bailey et al. (1996) J. Child. Psychol. Psychiatry, 37, 89-126).

Another disorder that causes ASD is a maternally inherited duplicationof 15q11-q13 (dup(15q)) (see, e.g. Folstein et al., (2001) Nat. Rev.Genet., 2, 943-955; Belmonte et al., (2004) Mol. Psychiatry, 9, 646-663;Veenstra-Vanderweele et al., (2004) Annu Rev Genomics Hum Genet, 5,379-405; Muhle et al., (2004) Pediatrics, 113, e472-486; and Sutcliffeet al. (2003) J. Am. Acad. Child. Adolesc. Psychiatry, 42, 253-256).Multiple repeat elements within the region mediate a variety ofrearrangements, including interstitial duplications, interstitialtriplications, and supernumerary isodicentric marker chromosomes (see,e.g. Wang et al. (2004) Am. J. Hum. Genet., 75, 267-281). Dup(15q)occurs with an estimated frequency of 1:600 in children withdevelopmental delay (see, e.g. Thomas et al. (2003) Am. J. Med. Genet.A, 120, 596-598) and is the most common copy number variation causingASD (see, e.g. Veenstra-Vanderweele et al., (2004) Annu Rev Genomics HumGenet, 5, 379-405; and Sutcliffe et al. (2003) J. Am. Acad. Child.Adolesc. Psychiatry, 42, 253-256). Over-expression of ubiquitin proteinligase E3A (UBE3A) and/or ATPase Class V type 10A (ATP10A) couldrepresent a major underlying molecular factor for autism (see, e.g.Herzing et al. (2002) Hum. Mol. Genet., 11, 1707-1718; and Herzing etal. (2001) Am. J. Hum. Genet., 68, 1501-1505). However, autism is not auniversal finding in maternal uniparental disomy of the 15q11-q13region, a condition in which UBE3A and ATP10A are over-expressed (see,e.g. Sutcliffe et al. (2003) J. Am. Acad. Child. Adolesc. Psychiatry,42, 253-256). These results suggest that dysregulation of non-imprintedgenes in the duplicated region and/or throughout the whole genome maycontribute to the autistic phenotype observed in dup(15q). Therefore, wereasoned that the identification of genes whose expression isdysregulated by both FMR1-FM and dup(15q) may provide genes relevant toASD, since the two genetic abnormalities represent cases where singlemutations, either a trinucleotide repeat or copy number variation (see,e.g. Sebat et al. (2004) Science, 305, 525-528), cause ASD. We alsowanted to examine, as a proof of principle, whether lymphoblast geneexpression profiles identified by microarrays can differentiate betweenthese single mutation “simple” causes of autism and controls. If thiswere the case, this would provide a basis for further study using thistechnique in idiopathic autism, where more multigenic inheritance andenvironmental influences may be at play (see, e.g. Folstein et al.,(2001) Nat. Rev. Genet., 2, 943-955; Belmonte et al., (2004) Mol.Psychiatry, 9, 646-663; Veenstra-Vanderweele et al., (2004) Annu RevGenomics Hum Genet, 5, 379-405; Muhle et al., (2004) Pediatrics, 113,e472-486).

Recently, several studies have suggested that lymphoblastoid cells canbe used to detect biologically plausible correlations between candidategenes and neuropsychiatric diseases, including Rett syndrome (see e.g.Horike et al. (2005) Nat. Genet., 37, 31-40), nonspecific X-linkedmental retardation (see, e.g. Meloni et al. (2002) Nat. Genet., 30,436-44), bipolar disorder (see, e.g. Iwamoto et al. (2004) Mol.Psychiatry, 9, 406-416), FXS (see e.g. Brown et al. (2001) Cell, 107,477-87) and dup(15q) (see, e.g. Baron et al. (2006) Hum. Mol. Genet, 15,853-869). In the present study, we investigated whether gene expressionprofiles of lymphoblastoid cells could be used (i) to differentiateautistic subjects who were ascertained and diagnosed as having ASD inthe Autism Genetic Resource Exchange (AGRE) (see, e.g. Geschwind et al.(2001) Am. J. Hum. Genet., 69, 463-466) repository into etiologicalcategories (FMR1-FM and dup(15q)) and (ii) to identify common genes andpathways that might be relevant to autism across these two distinctforms. Here, we demonstrate that the gene expression profile was able toclearly distinguish individuals based on their etiology. We alsoidentified 68 genes commonly dysregulated in autism with FMR1-FM anddup(15q). Interestingly, we identified a molecular connection betweenFMR1-FM and dup(15q), CYFIP1, which was significantly induced indup(15q) and is known to antagonize certain aspects of FMRP function(see, e.g. Schenck et al. (2003) Neuron, 38, 887-98). We furtherdemonstrated that the expression of janus kinase and microtubleinteracting protein 1 (JAKMIP1) and G protein-coupled receptor 155(GPR155) were commonly dysregulated by either reduction of FMR1 orinduction of CYFIP1 in vitro. The expression of JAKMIP1 was alsodysregulated in the brain of the FMR1 knock-out mouse. Finally, we wereable to show that JAKMIP1 and GPR155 were dysregulated in males withautism spectrum disorders (ASD), relative to their non-affectedsiblings, providing independent confirmation suggesting that these genesare associated with ASD.

Results

Hierarchical Clustering and Principal Component Analysis DistinguishIndividuals Based on Genetic Etiology

We analyzed the whole-genome mRNA expression profile in lymphoblastoidcells from 15 autistic males (8 autistic males with FMR1-FM and 7autistic males with dup(15q)) and 15 non-autistic control males fromAGRE (see supplemental table S1 in Nishimura et al., Human MolecularGenetics 2007 16(14): 1682-1698, the contents of which are incorporatedherein by reference) using Agilent Whole Genome Human Microarrays.Overall, out of 41,000 probes analyzed, 31,044 probes, representing23,822 genes, were expressed in the lymphoblastoid cells. To find genesthat were differentially expressed across the three subject groups, theexpression profile of the lymphoblastoid cells was subjected to Analysisof Variance (ANOVA) (see, e.g. Cui et al. (2003) Genome Biol., 4, 210).ANOVA identified 293 probes (277 genes) below a defined false discoveryrate (FDR) threshold of 5% (see supplemental table S2 in Nishimura etal., Human Molecular Genetics 2007 16(14): 1682-1698, the contents ofwhich are incorporated herein by reference). It has been shown that theexpression of FMR1 is decreased in lymphoblastoid cells with FMR1-FM(see, e.g. Sutcliffe et al. (1992) Hum. Mol. Genet., 1, 397-400) andthat the expression of UBE3A is increased in lymphoblastoid cells withdup(15q) (see, e.g. Herzing et al. (2002) Hum. Mol. Genet., 11,1707-1718). Concordant with these reports, FMR1 and UBE3A were among the293 differentially expressed probes, providing independent controls forthe microarray analysis.

As shown in FIGS. 1A and B, hierarchical clustering using the 293 probesclearly classified individuals based on their genotype. The 293 probeswere also subjected to principal component analysis (PCA). As shown inFIG. 1C, 3 dominant PCA components that contained 70% of the variance inthe data matrix clearly separated individuals based on genetic etiology.In this plot, the first principal component axis accounted for 56% ofthe variance in the data set and clearly separated autism with FMR1-FMand dup(15q) from controls, whereas the second principal component (PC2)accounted for 10% of the variance and segregated autism with FMR1-FMfrom autism with dup(15q). The top 10 genes contributing to PC2 includeFMR1, UBE3A, CYFIP1, non-imprinted in Prader-Willi/Angelman syndrome 2(NIPA2), and hect domain and RLD 2 (HERC2). The latter four genes areall located within the 15q11-q13 region. These results suggest that theselective reduction of FMR1 and the selective induction of the fourgenes located in 15q11-q13 differentiated autism with FMR1-FM fromautism with dup(15q). These data provide a critical proof of principlethat the gene expression profile of lymphoblastoid cells could be usedto subgroup subjects with autism based on their genetic etiologies whenthe etiologies are due to a single mutation or copy number variation.

Microarray Analyses Revealed the Significant Overlap of FMR1-FM anddup(15q)

To identify the set of the most robustly differentially expressed genesin each group, we identified genes found using three differentstatistical methods, ANOVA, Significant Analysis of Microarray (SAM)(see, e.g. Tusher et al. (2001) Proc. Natl. Acad. Sci. USA, 98,5116-5121) and Rank Product Analysis (RankProd) (see, e.g. Breitling etal. (2004) FEBS Lett., 573, 83-92). SAM is a modified t-test statistic,whereas RankProd is a non-parametric statistic that detects items thatare consistently highly ranked in a number of lists. SAM identified 5139probes and 1281 probes as significant (FDR<5%) in autism with FMR1-FMand dup(15q), respectively (FIGS. 2A and B). RankProd identified 2281probes and 1444 probes as significant (FDR<5%) in autism with FMR1-FMand dup(15q), respectively (FIGS. 2A and B). The combination of ANOVA,SAM and RankProd identified 146 probes (120 genes) in autism withFMR1-FM and 97 probes (80 genes) in autism with dup(15q) (FIG. 2C).Eighty-three probes representing 68 genes were dysregulated in bothautism with FMR1-FM and dup(15q) (see Table 1 in Nishimura et al., HumanMolecular Genetics 2007 16(14): 1682-1698, the contents of which areincorporated herein by reference). This degree of overlap was highlysignificant (hypergeometric probability, P=1.2×10-153). Fifty-two genesand 12 genes were selectively dysregulated in either autism with FMR1-FMand autism with dup(15q), respectively (see Table 2 in Nishimura et al.,Human Molecular Genetics 2007 16(14): 1682-1698, the contents of whichare incorporated herein by reference).

qRTPCR Confirmed the Differential Expression Identified by theMicroarray Analysis

To validate the differential expression identified by microarrayanalysis using independent methods, we performed quantitative real-timePCR analysis (qRTPCR) of 19 genes chosen as a cross section using thesame samples used in the microarray analysis. qRTPCR confirmed that 17of the 19 genes were differentially expressed as expected by themicroarray analysis (FIG. 3, A-C). There was an overall highlysignificant correlation between microarray and qRTPCR results (Pearsoncorrelation, r=0.57, P<0.0001).

CYFIP1 was one of the genes selectively induced in autism with dup(15q).Because CYFIP1 is known to antagonize FMRP (see, e.g. Schenck et al.(2003) Neuron, 38, 887-98), we reasoned that the induction of CYFIP1 indup(15q) might explain some of the significant overlap between autismwith FMR1-FM and dup(15q). JAKMIP1, also known as Marlin-1, wassignificantly induced in autism with FMR1-FM and had a positive trend inautism with dup(15q) (P=0.062), suggesting that JAKMIP1 could representa commonly dysregulated pathway. In fact, RankProd identified JAKMIP1 asa significantly up-regulated gene in dup(15q) by microarray analysis.This gene is a particularly biologically important candidate, given itsputative role in GABAB receptor expression (see, e.g. Couve et al.(2004) J. Biol. Chem., 279, 13934-13943) and the microtubule network(see, e.g. Steindler et al. (2004) J. Biol. Chem., 279, 43168-43177).

Functional Annotation Revealed Pathway Dysregulation

In an attempt to uncover the common functional meanings among thedifferentially expressed genes, we classified genes into gene ontologygroups using DAVID (see, e.g. Dennis et al. (2003) Genome Biol., 4, P3).Table 3 in Nishimura et al., Human Molecular Genetics 2007 16(14):1682-1698, the contents of which are incorporated herein by reference,shows the top 3 clusters identified by DAVID using the 68 genesdysregulated in autism with FMR1-FM and dup(15q), or the 52 genesselectively dysregulated in autism with FMR1-FM. The number of genesselectively dysregulated in autism with dup(15q) was too small toanalyze using the functional annotation clustering.

Genes related to cell communication (P=7.6×10-6) and signal transduction(P=2.2×10-5) were most significantly enriched in the 68 genes commonlydysregulated in autism with FMR1-FM and dup(15q). Genes related toimmune response (P=3.7×10-3) and defense response (P=7.3×10-3) were alsoenriched in this gene set. Genes related to chaperone (P=2.6×10-2) andprotein folding (P=3.2×10-2) were enriched in the 52 genes selectivelydysregulated in autism with FMR1-FM. Genes related to RNA binding(P=1.2×10-2) and mRNA metabolism (P=2.1×10-2) were also enriched in thisgene set, consistent with the FMRP protein's function as RNA bindingprotein important in regulatory translation (see, e.g. Bagni et al.(2005) Nat. Rev. Neurosci., 6, 376-387). Chaperones and folding proteinsare commonly found to operate co-translationally, providing a potentiallink with FMRP function.

To provide a more refined functional classification of genes, we usedIngenuity Pathway Analysis (IPA) (see, e.g. Ingenuity Pathway Analysis.(http://www.ingenuity.com/)), a powerful tool for investigating thebiological pathways represented by the genes commonly dysregulated inautism with FMR1-FM and dup(15q). IPA uses known protein-protein andgene-gene interactions that have been culled into a curated database andassociates the list of differentially expressed genes with biologicalnetworks. IPA identified three statistically significant networks, eachcontaining at least ten genes (see Table 4 and supplemental FIG. 1) inNishimura et al., Human Molecular Genetics 2007 16(14): 1682-1698, thecontents of which are incorporated herein by reference). Principalfunctions associated with these networks were cell cycle (P=5.2×10-8),cellular movement (P=1.3×10-8) and cell-to-cell signaling andinteraction (P=4.3×10-8). The “cell-to-cell signaling and interaction”was consistent with “cell communication” and “signal transduction”categories identified by the DAVID. The identification of the “moleculartransport” pathway containing JAKMIP1 was particularly salient, giventhis gene's known role in GABAR trafficking within neurons. There werealso other important genes in this pathway, including PSCD3, anADP-ribosylation factor of unknown CNS function and ACTN1, acytoskeletal anchoring protein. Based on this analysis, it is plausiblethat JAKMIP1 may act along with these genes in the segregation ofsignaling complexes involved in neural transmission.

Effect of Down-Regulation of FMR1 and Up-Regulation of CYFIP1 in aNeuronal Cell on the Expression of the Dysregulated Genes Identified inLymphoblastoid Cells

Although we identified dysregulated genes in autism with FMR1-FM anddup(15q) using lymphoblastoid cells, we were interested in whether theexpression of these genes would also be dependent on FMR1 and CYFIP1 inneuronal cells. To examine the effect of FMR1 and CYFIP1 in neuronalcells, we used the well characterized human neuronal cell line SH-SY5Y(see, e.g. Millar et al. (2005) Science, 310, 1187-1191). FMR1 andCYFIP1 dependence in SH-SY5Y cells were assessed using short hairpin RNA(shRNA) to reduce the expression of FMR1 and a plasmid expression vectorto induce the expression of CYFIP1, respectively. As shown in FIG. 4A,the expression of FMR1 was reduced to about 60% of its normal level inSH-SY5Y cells stably expressing FMR1 shRNAs, whereas the expression ofCYFIP1 was significantly induced (11-fold) in SH-SY5Y cells stablytransfected with the CYFIP1 plasmid.

We were able to further demonstrate the effect of down-regulation ofFMR1 and up-regulation of CYFIP1 on the expression of two key downstreamgenes (FIG. 4B). In SH-SY5Y cells transfected with FMR1 shRNA, theexpression of JAKMIP1 and GPR155 were significantly reduced and induced,respectively. In SH-SY5Y cells over-expressing CYFIP1, the expression ofJAKMIP1 and GPR155 were also reduced and induced, respectively. Thesefindings demonstrated that the expression of JAKMIP1 and GPR155 werealso dependent on FMR1 and CYFIP1 in SH-SY5Y cells and that reduction ofFMR1 and induction of CYFIP1 can share common downstream effects on theexpression of JAKMIP1 and GPR155.

The Expression of JAKMIP1 Protein was Dependent on FMR1 and CYFIP1

Then, we validated the effect of FMR1 or CYFIP1 on the proteinexpression of JAKMIP1 in the central nervous system (CNS). We examinedthe expression of the JAKMIP1 protein in the cortex of FMR1 knock-out(KO) and wild-type (WT) mice and SH-SY5Y cells transfected with theCYFIP1 over-expression plasmid. The expression of JAKMIP1 protein wasreduced in cortex of FMR1 KO mice (FIG. 5A) and SH-SY5Y cellsover-expressing CYFIP1 (FIG. 5B). These findings confirmed the in vitrofindings that the expression of JAKMIP1 was dependent on FMR1 in mousebrain, suggesting that at least some of the changes observed inlymphoblastoid cells reflect similar changes in the CNS.

The Expression of JAKMIP1 and GPR155 were Significantly DifferentBetween 27 Male Sib Pairs Discordant for Idiopathic ASD

To determine the potential generalizability of these findings toidiopathic autism, we examined whether the expression of JAKMIP1 andGPR155 were also dysregulated in lymphoblastoid cells from idiopathicASD cases. We selected 27 male sib pairs discordant for ASD from AGRE(see supplemental table S1 in Nishimura et al., Human Molecular Genetics2007 16(14): 1682-1698, the contents of which are incorporated herein byreference). The 27 males with ASD did not have FMR1-FM or dup(15q) andhad surrogate IQ markers (Raven's progressive matrices) >70. As shown inFIG. 6, the expression of JAKMIP1 and GPR155 were significantlydysregulated in the 27 males with ASD compared to their sibs withoutASD. These results show that the dysregulation of JAKMIP1 and GPR155 areassociated with ASD. The lack of general intellectual disability in thisASD group also shows that these dysregulation are not simply due to anon-specific cognitive impairment or intellectual disability observed inFXS and dup(15q). However, both in vitro (SH-SY5Y cells) and in vivo(brain) CNS tissues, the direction of JAKMIP1 and GPR155 regulation wereopposite to that observed in lymphoblastoid cells. The differences mayreflect many facts, including immortalization or alternative regulatorysignaling pathways in different tissues. However, these data areconsistent between FMR1-FM and dup(15q) and indicate that expression ofJAKMIP1 and GPR155 are regulated by both FMR1 and CYFIP1 levels, albeitdifferently between neural tissues and lymphoblastoid cells, providingpotential common signaling pathways dysregulated in ASD.

In this study, we performed global mRNA expression profiling from maleswith autism carrying either FMR1-FM or dup(15q) and control males. Wefound that these autistic individuals can be differentiated based ontheir genetic etiologies by lymphoblast gene expression profiles.Interestingly, this analysis also revealed a common gene expressionsignature across these two distinct genetic conditions leading to ASDthat was significantly different from control profiles. We used theintersection of three different statistical tests to identify the mostrobustly differentially expressed genes (see, e.g. Cui et al. (2003)Genome Biol., 4, 210; Tusher et al. (2001) Proc. Natl. Acad. Sci. USA,98, 5116-5121; and Breitling et al. (2004) FEBS Lett., 573, 83-92). TheqRTPCR data confirmed this gene selection strategy.

Gene Expression Profiles of Lymphoblastoid Cells Carrying the FMR1-FM

We identified 120 genes differentially expressed in FMR1-FM carrierscompared with controls. Among these genes, NR3C1 and VIM were previouslyidentified as target RNAs of FMRP (see, e.g. Miyashiro et al. (2003)Neuron, 37, 417-431), although the mRNA expression changes of thesegenes in FMR1-FM have not been reported.

Brown et al (see, e.g. Brown et al. (2001) Cell, 107, 477-87) previouslyidentified 144 genes as differentially expressed in lymphoblasts withFMR1-FM using pooled fragile X lymphoblastoid cells and pooled normallymphoblastoid cells. Because there was no overlap except for FMR1between these 144 genes and the 120 genes identified here with our moststringent analyses using ANOVA, SAM and RankProd, we used the largergene list identified by either SAM and/or RankProd to compare with the144 genes identified by Brown et al. We found that 13 genes were sharedin these gene lists, including iduronate 2-sulfatase (IDS), hairy andenhancer of split 1 (HES1) and immunoglobulin superfamily, member 3(IGSF3) as up-regulated genes and CDK2-associated protein 2 (CDK2AP2),ubiquitin specific peptidase 8 (USP8), MAX-like protein X (MLX),ribosomal protein S5 (RPS5), C-terminal binding protein 1 (CTBP1),spleen tyrosine kinase (SYK), F-box protein 6 (FBXO6), mitogen-activatedprotein kinase kinase kinase 11 (MAP3K11), sorting nexin 15 (SNX15) andCD44 antigen (CD44) as down-regulated genes. Although these genes havenot been reported as associated with FMR1 or autism, HES1 was associatedwith attention-deficit hyperactive disorder (ADHD) (see, e.g. Brookes etal. (2006) Mol. Psychiatry, 10, 934-953), which is a symptom frequentlyseen in FXS (50) and overlapping with ASD (see, e.g. Todd, R. D. (2001)Child Adolesc. Psychiatr. Clin. N. Am., 10, 209-24). The relatively lowoverlap between the two gene lists could be due to the difference ofclinical features of individuals (autism vs. not specific for autism),experimental design (each individual vs. pooled), microarray platforms(Agilent vs. Affymetrix) and the statistical analysis used to find thedifferential expression between groups. The initial study (see, e.g.Brown et al. (2001) Cell, 107, 477-87) whose primary aim was to findFMRP ligand mRNPs was relatively underpowered to detect overalldifferences in gene expression and our study used very conservativestatistical criteria. However, this core set of genes provides aninteresting gene list for further investigation.

Gene Expression Profiles of Lymphoblastoid Cells with dup(15q)

We identified 80 genes differentially expressed in dup(15q) carrierscompared with controls. Among these genes, 4 genes located in 15q11-q13(the region of duplication) UBE3A, CYFIP1, NIPA2, and GOLGA8F were allinduced. It is important to note that 5 other genes located in theduplicated region, tubulin gamma complex associated protein 5 (TUBGCP5),HERC2, HERC2 pseudogene 2 (HERC2P2), NIPA1, and ATP10A and were alsoidentified as up-regulated genes by at least one of the three differentstatistical analyses (see supplemental table S3 in Nishimura et al.,Human Molecular Genetics 2007 16(14): 1682-1698, the contents of whichare incorporated herein by reference). Four other genes in theduplicated region, gamma-aminobutyric acid A receptor (GABR) beta 3(GABRB3), GABR alpha 5 (GABRA5), GABR gamma 3 (GABRG3), oculocutaneousalbinism II (OCA2) and necdin homolog (NDN), were not expressed atdetectable levels in the lymphoblastoid cells. It is important toemphasize that the 15q11-q13 region is subject to paternal imprinting.Three paternally imprinted genes, makorin ring finger protein 3 (MKRN3),MAGE-like 2 (MAGEL2) and SNRPN upstream reading frame (SNURF)-smallnuclear ribonucleoprotein polypeptide N (SNRPN) were expressed in thelymphoblasts, but showed no significant changes relative to controls.This data is consistent with the fact that the duplicated region wasmaternally derived in all 7 cases analyzed in this study. So, overall,these findings suggest that the genes located in the duplicated regionwere globally upregulated except for the paternally imprinted genes.Global up-regulation due to gene-dosage has also been reported in Downsyndrome (see, e.g. Tang et al. (2004) Ann. Neurol., 56, 808-814; andMao et al. (2003) Genomics, 81, 457-467).

Baron et al (see, e.g. Baron et al. (2006) Hum. Mol. Genet, 15, 853-869)identified 81 known genes as differentially expressed in lymphoblastoidcells with dup(15q) (7 individuals) compared to controls (8 individuals)using the Affymetrix platform. They identified upregulation of UBE3A,NIPA1, NIPA2 and HERC2, consistent with our results. We used the genelist identified by SAM and/or RankProd to compare with the 81 genesidentified by Baron et al and identified 11 other genes shared in thetwo gene lists, a significant overlap (the hypergeometric probability is0.001). These genes were abhydrolase domain containing 6 (ABHD6),potassium channel, subfamily K, member 1 (KCNK1), hypothetical proteinKIAA1147, and zinc finger, DHHC domain containing 14 (ZDHCC14) asup-regulated and Rho GTPase activating protein 25 (ARHGAP25), cloneLOC387882, leukotriene B4 12-hydroxydehydrogenase (LTB4DH), cloneMGC27165, PFTAIRE protein kinase 1 (PFTK1), zinc finger protein 43(ZNF43) and ring finger protein 41 (RNF41) as down-regulated. Therelationships between these genes and autism remain unknown. Again, asis the case of FMR1-FM, these genes represent a set of independentlyreplicated genes between two studies.

Significant Overlap of Dysregulated Genes in Autism with FMR1-FM anddup(15q)

We identified 68 genes that were dysregulated in both autism withFMR1-FM and dup(15q), a very significant result (hypergeometricprobability of this overlap is 1.2×10-153). However, we can not formallyexclude the possibility that some of the 68 common dysregulated genesmight be related to common pathways between FMR-FM and dup(15q)unrelated to ASD. Microarray analysis using lymphoblastoid cells withFMR1-FM or dup(15q), but without ASD is needed to exclude thepossibility, as was done in Tuberous Sclerosis cases with and withoutautism (see, e.g. Tang et al. (2004) Ann. Neurol., 56, 808-814).

We found that the expression of CYFIP1 was significantly induced inautism with dup(15q). CYFIP1 protein has been shown to antagonize FMRPin the eye and nervous system of Drosophila (see, e.g. Schenck et al.(2003) Neuron, 38, 887-98). In FXS, the absence of FMRP, a bindingpartner to CYFIP1, results in excess free CYFIP1 protein. Similarly,excess free CYFIP1 protein may be the outcome of dup(15q). Thus,antagonization of FMRP by over-expression of CYFIP1 protein, and/oralternate actions of excess CYFIP1 protein may be common mechanisticlinks between FMR1-FM and dup(15q).

Effect of FMR1 and CYFIP1 on the Commonly Dysregulated Genes in SH-SY5Yand Mouse Brain

We validated the effect of down-regulation of FMR1 in SH-SY5Y cells andmouse brain and up-regulation of CYFIP1 in SH-SY5Y cells on theexpression of the commonly dysregulated genes identified in patientlymphoblastoid cells. We demonstrated that the expression of JAKIMIP1and GPR155 were dysregulated by reduction of FMR1 and induction ofCYFIP1 in SH-SY5Y cells. JAKMIP1 protein was also dysregulated byknock-out of FMR1 in mouse brain. Interestingly, the direction ofchanges observed in both of these genes is opposite in neural tissues(SH-SY5Y cells and brain) and lymphoblastoid cells. Such differencesbetween brain and blood cells have been previously observed in othersignaling pathways (see, e.g. Iwamoto et al. (2004) Mol. Psychiatry, 9,406-416; and Middleton et al. (2005) Am. J. Med. Genet. BNeuropsychiatr. Genet., 136, 12-25). It is likely that it is not theprecise direction observed in lymphoblastoid cells that is mostimportant, but the common dysregulation of JAKMIP1 and GPR155,downstream of these single gene defects and which is observed inidiopathic ASD.

JAKMIP1 is associated with janus kinases (see, e.g. Steindler et al.(2004) J. Biol. Chem., 279, 43168-43177), microtubles (see, e.g.Steindler et al. (2004) J. Biol. Chem., 279, 43168-43177) and GABRBreceptors (see, e.g. Couve et al. (2004) J. Biol. Chem., 279,13934-13943). The expression levels of JAKMIP1 affect the intracellularlevels of the GABRB receptor (see, e.g. Couve et al. (2004) J. Biol.Chem., 279, 13934-13943). Because the GABRB receptor could interact withthe metabotropic glutamate receptor 1 (mGluR1) and increase theglutamate sensitivity of mGluR1 (see, e.g. Tabata et al. (2004) Proc.Natl. Acad. Sci. USA, 101, 16952-16957), JAKMIP1 might affect mGluR1signaling through GABRB receptors. It is important to note that mGluRsignaling is exaggerated in FMR1 knock-out mouse (see, e.g. Bear et al.(2004) Trends. Neurosci., 27, 370-377) and that glutamergic andGABAergic system have been reported to be abnormal in autism (see, e.g.Polleux et al. (2004) Ment. Retard. Dev. Disabil. Res. Rev., 10,303-317). JAKMIP1 is highly expressed throughout the mouse brain,especially in hippocampus where GABRB receptors and mGluR1 are alsohighly expressed (see, e.g. Allen Brain Atlas.(http://www.brain-map.org/)). Although the function of GPR155 isunknown, it is highly expressed in the limbic system in mouse brain(see, e.g. Allen Brain Atlas. (http://www.brain-map.org/)), suggestingthat GPR155 might have functions relevant to the limbic system.

It is also interesting to consider how the reduction of FMR1 and theinduction of CYFIP1 might regulate the expression of JAKMIP1 and GPR155.G-quadruplex motifs in RNA have been shown to play significant roles inFMRP binding (see, e.g. Darnell et al. (2001) Cell, 107, 489-99). UsingQGRS mapper (see, e.g. Kikin et al. (2006) Nucleic Acids Res., 34,W676-682), we found that human and mouse JAKMIP1 each had two of theG-quadruplex (G2N2-4G2N2-4G2N2-4G2) and that human and mouse GPR155 hadfive and one of the G-quadruplex, respectively. FMRP can also bindtarget RNAs through non-coding RNAs (see, e.g. Zalfa et al. (2003) Cell,112, 317-327) or microRNAs (see, e.g. Jin et al. (2004) Nat. Cell.Biol., 6, 1048-1053). Using miRBase (see, e.g. Griffiths-Jones et al.(2006) Nucleic Acids Res., 34, D140-144), we found putative microRNAbinding sites in human and mouse JAKIMIP1 and GPR155. Further studiesare required to clarify the functional importance of JAKMIP1 and GPR155in autism and the mechanism of regulation of these genes by FMR1 andCYFIP1. In this regard, the potential link with neuronal transmission isintriguing.

The Expression of JAKMIP1 and GPR155 were also Dysregulated in 27 Maleswith Idiopathic ASD

The findings in autism with FMR1-FM and dup(15q) suggest that JAKMIP1and GPR155 may be involved more generally in idiopathic ASD, since theirdysregulation is observed in neural cells and brain. We tested whetherdysregulation of these genes were more generalizable in an independentsample consisting of idiopathic ASD cases. To attempt to reduce theheterogeneity of idiopathic ASD and extend these findings beyond thosewith mental retardation or intellectual disability, we used an IQsurrogate based on Raven's Progressive Matrices, which is highlycorrelated with IQ defined by other measures (see, e.g. Mottron, L.(2004) J. Autism Dev. Disord., 34, 19-27). We selected 27 ASD males withan IQ score over 70. These data demonstrated that the expression ofJAKMIP1 and GPR155 are significantly dysregulated in lymphoblastoidcells from idiopathic ASD compared to controls. These results based onindependent data on lymphoblastoid cell gene expression from ASDsubjects with FMR1-FM, or dup(15q), as well as idiopathic ASD suggestthat JAKMIP1 and GPR155 may be useful as biomarkers for ASD.

The mechanism for the opposite regulation of JAKMIP1 and GPR155 inlymphobastoid cells and neural cells remain to be elucidated. There areseveral previous reports of genes showing the opposite expressionbetween lymphoblastoid cells and brains in neuropsychiatric disease. Oneexample is inositol monophosphatase 2 (IMPA2) that has been identifiedas a plausible locus for bipolar disorder (see, e.g. Yoshikawa et al.(1997) Mol. Psychiatry, 2, 393-397; Nothen et al. (1999) Mol.Psychiatry, 4, 76-84; and Lin et al. (2005) Am. J. Hum. Genet., 77,545-555). The expression of IMPA2 was reduced and induced inlymphoblastoid cells and brains, respectively, in patients with patientswith bipolar disorder (see, e.g. Yoon et al. (2001) Mol. Psychiatry, 6,678-683). A genetic association between IMPA2 promoter polymorphism andbipolar disorder has been confirmed (see, e.g. Sjoholt et al. (2004)Mol. Psychiatry, 9, 621-629; and Ohnishi et al. (2007)Neuropsychopharmacology). In this regard, it is notable that GPR155 islocated on 2q31.1, 300 kb from D2S2188, which has shown strong linkageto autism in studies by two independent groups (see, e.g. IMGSAC (2001)Am. J. Hum. Genet., 69, 570-581; and Romano et al. (2005) Psychiatr.Genet., 15, 149-150). Association analysis for GPR155 and JAKMIP1 areongoing using the large AGRE cohort. These data provide the firstidentification and independent validation of the potential roles ofJAKMIP1 and GPR155 dysregulation in ASD. Further work is needed tounderstand the functional consequences of these changes in thedeveloping brain, and to assess the general utility of these and othergenes as potential biomarkers.

Materials and Methods

Individuals and Lymphoblastoid Cells Analyzed in this Study

We have analyzed individuals diagnosed with ASD using standard validatedmeasures, including the Autism Diagnostic Interview (ADI-R) (see, e.g.Lord et al. (1994) J. Autism Dev. Disord., 24, 659-685) and AutismDiagnostic Observation Schedule (ADOS) (see, e.g. Lord et al. (2001) Am.J. Med. Genet., 105, 36-38). Eight males with FMR1-FM and 3 males withdup(15q) were drawn from AGRE (see, e.g. Geschwind et al. (2001) Am. J.Hum. Genet., 69, 463-466) (http://www.agre.org/). An additional 4 maleswith dup(15q) were obtained from NIGMS Human Genetic Cell Repository. 27males without autism, FMR1-FM and dup(15q) were drawn from the AGRE forcontrols. In addition, another 27 males with idiopathic ASD who hadunaffected male siblings were chosen from AGRE for a comparison sample(see supplemental table S1 in Nishimura et al., Human Molecular Genetics2007 16(14): 1682-1698, the contents of which are incorporated herein byreference). Surrogate IQ scores (using the Raven Progressive Matrices)were available. FMR1-FM and dup(15q) were examined by PCR andfluorescence in situ hybridization, respectively. The 15q11-q13duplicated region in the 7 males analyzed in this study were allmaternally derived. We also used 14 other individuals from AGRE forcommon reference (pool) in microarray analysis. Lymphoblastoid celllines (human Epstein-Barr virus transformed lymphocytes) from theseindividuals were available from AGRE and NIGMS cell repositories.

The lymphoblastoid cells of the subjects were grown in RPMI 1640 mediumwith 2 mM L-glutamine and 25 mM HEPES (Invitrogen, Carlsbad, Calif.,USA), 10% fetal bovine serum, 1× Antibiotic-Antimycotic solution(Invitrogen, Carlsbad, Calif., USA) at 37° C. in a humidified 5% CO2Chamber. Cells were grown to a density of 6×10⁵/ml. Special attentionwas given to maintain all the cell lines in the same conditions tominimize environmental variation.

Microarray Experiment

A total of 9×10⁶ of lymphoblastoid cells were seeded out in a T-75 flaskin 30 ml of fresh medium. After 24 hours, total RNA was extracted fromthe cells using RNeasy Mini Kit with DNase treatment (Qiagen, Valencia,Calif., USA) according to manufacturer's protocol. The RNA quantity andquality was measured by ND-100 (Nanodrop, Wilmington, Del., USA) and2100 Bioanalyzer (Agilent, Santa Clara, Calif., USA), respectively.

Target preparation was performed using Low RNA Input Fluorescent LinearAmplification Kit (Agilent, Santa Clara, Calif., USA) according to themanufacturer's protocol. We extracted total RNA from lymphoblastoidcells from each individual and made target and labeled it with Cy5fluorescence. We also made reference target by using pooled total RNAfrom the 14 individuals for reference and labeled it with Cy3fluorescence. The generated targets were mixed and subjected tohybridization to the Whole Human Genome Array G4112A (Agilent, SantaClara, Calif., USA) according to the manufacturer's protocol. Scanningof the microarrays were done by DNA microarray scanner (Agilent, SantaClara, Calif., USA).

Scanner output image files were normalized and filtered by using FeatureExtraction Software v8.5 (Agilent, Santa Clara, Calif., USA).Normalization was performed so that overall intensity ratio of Cy5 toCy3 was equal to one. Probes with signal to noise ratio >2.7 in both Cy3and Cy5 in at least 14 of 15 controls were used for further analysis.

Statistical Analysis of Microarray Data

ANOVA was performed by MeV3.1 (see, e.g. Saeed et al. (2003)Biotechniques, 34, 374-378). P value was calculated based on 1000permutation. Hierarchical clustering using Spearman's rank correlationwith average linkage clustering were performed by MeV3.1 Principalcomponent analysis was performed by GeneSpring GX7.3 (Agilent, SantaClara, Calif., USA). SAM (see, e.g. Tusher et al. (2001) Proc. Natl.Acad. Sci. USA, 98, 5116-5121) and RankProd (see, e.g. Breitling et al.(2004) FEBS Lett., 573, 83-92) were performed using Bioconductor (see,e.g. Gentleman et al. (2004) Genome Biol., 5, R80) packages Siggene andRankProd, respectively. 100 and 1000 permutation were performed forcross-validation in SAM and RankProd, respectively. We used threedifferent statistical tests to conservatively identify the most robustlydifferentially expressed genes. Numerous feature selection methods havebeen applied to the identification of differentially expressed genes inmicroarray data (see, e.g. Jeffery et al. (2006) BMC Bioinformatics, 7,359). The genes commonly identified by ANOVA, SAM and RankProd arelikely to be differentially expressed, given the relative robustness ofthese statistical approaches (see, e.g. Cui et al. (2003) Genome Biol.,4, 210; Tusher et al. (2001) Proc. Natl. Acad. Sci. USA, 98, 5116-5121;Breitling et al. (2004) FEBS Lett., 573, 83-92; and Jeffery et al.(2006) BMC Bioinformatics, 7, 359). Functional Annotation Clustering wasperformed by DAVID (see, e.g. Dennis et al. (2003) Genome Biol., 4, P3)with medium classification stringency. The clustering algorithm is basedon the hypothesis that similar annotations should have similar genemembers. The Functional Annotation Clustering uses two differentstatistics to measure the degree of the common genes between twoannotations and to classify the groups with similar annotations. TheGroup Enrichment Score is the geometric mean (in −log scale) of amember's p-values in a corresponding annotation cluster. IPA was used tofind significant pathways related to the genes commonly dysregulated inautism with FMR1-FM and dup(15q). The Ingenuity Pathway Knowledge Basebuilds gene networks based upon known protein and gene interactions(see, e.g. Ingenuity Pathway Analysis, e.g. by searching“www.ingenuity.com”). IPA determines a statistical score for eachnetwork according to the probability of the network given the gene list.The Ingenuity Pathway Knowledge Base provides pathways with biologicalfunction based upon the scientific literature. The significance valueassociated with Functions and Pathways measures how likely it is thatgenes from the dataset file participate in that biological function. Thesignificance was expressed as a p-value, which is calculated using theright-tailed Fisher's Exact Test.

Quantitative Real Time PCR Analysis (qRTPCR)

Total RNAs was used to make cDNA by SuperScript III First-StrandSynthesis SuperMix (Invitrogen, Carlsbad, Calif., USA). qRTPCR was doneby ABI Prism 7900 using Platinum SYBR Green qPCR SuperMix UDG with ROX(Invitrogen, Carlsbad, Calif., USA). Thermal cycling consisted on aninitial step at 50° C. for 2 min followed by another step at 95° C. for2 min and 50 cycles of 95° C. for 15 sec and 60° C. for 30 sec. qRTPCRwas performed for 16 genes. The primers used in this study are shown inSupplementary table S4 in Nishimura et al., Human Molecular Genetics2007 16(14): 1682-1698, the contents of which are incorporated herein byreference. TaqMan probe (Hs00327005_m1, Applied Biosystems, Foster City,Calif., USA) was used to measure JAKMIP1 expression in lymphoblastoidcells. Data was normalized by the quantity of hypoxanthinephosphoribosyltransferase 1 (HPRT1). HPRT1 was selected rather thanbeta-actin, glyceraldehyde-3-phosphate dehydrogenase or other possibleinternal controls because it was shown to be most stable RNA speciesfrom the lymphoblastoid cell lines. This allowed us to account for thevariability in the initial template concentration as well as theconversion efficiency of the reverse transcription reaction.

Transfection of shRNA

To construct retrovirus vectors expressing shRNAs, oligonucleotidesencoding stem-loop shRNAs for FMR1 (see supplementary table S4 inNishimura et al., Human Molecular Genetics 2007 16(14): 1682-1698, thecontents of which are incorporated herein by reference) and negativecontrol were ligated into the BamHI and EcoRI site of the pSIREN-RetroQ(BD Clontech, Mountain View, Calif., USA). PT67 cells (BD Clontech,Mountain View, Calif., USA) were transfected for retrovirus production.A total of 6×106 of SH-SY5Y cells were seeded out in a T-75 flask in 20ml of fresh medium of DMEM (Invitrogen, Carlsbad, Calif., USA) with 10%FBS. After 1 day, SH-SY5Y cells were infected with retroviruses in thepresence of 5 μg/ml of polybrene. After 2 days, the SH-SY5Y cells weretreated with 10 μg/ml of puromycin (Sigma, St. Louis, Mo., USA). Cellsthat survived after 4 weeks were collected and this population of cellswas used for further experiments. Total RNA were extracted from thecells using RNeasy Mini Kit with DNase treatment (Qiagen, Valencia,Calif., USA) according to the manufacturer's protocol. We comparedSH-SY5Y cells expressing FMR1 shRNA (n=4) and SH-SY5Y cells expressingshRNAs for negative control (n=4) to examine the effect of reduction ofFMR1 on the expression of JAKMIP1 and GPR155.

Transfection of CYFIP1

The human CYFIP1 coding region (aa 1-1254) obtained by PCR using IMAGEclone 10625411 (ATCC, Manassas, Va., USA) was subcloned into the EcoRVand NotI sites of the plasmid vector pIRES-neo3 (BD Clontech, MountainView, Calif., USA). The sequence of the construct was confirmed byautomated DNA sequencing.

A total of 6×106 of SH-SY5Y cells were seeded out in a T-75 flask in 20ml of fresh medium of DMEM (Invitrogen, Carlsbad, Calif., USA) with 10%FBS. After 1 day, SH-SY5Y cells were transfected with 120 ul oflipofectamine 2000 (Invitrogen, Carlsbad, Calif., USA) diluted with 3 mlof OptiMEM (Invitrogen, Carlsbad, Calif., USA) and 24 g of plasmid(pIRES-CYFIP1 or pIRES) diluted with 3 ml of OptiMEM (Invitrogen,Carlsbad, Calif., USA). After 5 min at room temperature, they werecombined and incubated for 20 min. The reaction mixture was added with16 ml of DMEM with 10% FBS. The cell culture medium was replaced by thissolution. After 2 days, the SH-SY5Y cells were treated with 500 μg/ml ofG418 (Invitrogen, Carlsbad, Calif., USA). Cells that survived after 3weeks were collected and this population of cells was used for furtherexperiments. Total RNA was extracted from the cells using RNeasy MiniKit with DNase treatment (Qiagen, Valencia, Calif., USA) according tothe manufacturer's protocol. We compared SH-SY5Y cells stablytransfected with expression vector for CYFIP1 (n=7) and SH-SY5Y cellstransfected with empty expression vector (n=8) to examine the effect ofinduction of CYFIP1 on the expression of JAKMIP1 and GPR155. Protein wasalso extracted using Cellytic M (Sigma, St. Louis, Mo., USA) withproteinase inhibitors (Sigma, St. Louis, Mo., USA) according to themanufacturer's protocol.Animals and Tissue Collection

Wild-type (WT) and FMR1 KO mice were raised at the Emory Universityanimal facility and treated in accordance with National Institute ofHealth regulations and under approval of the Emory UniversityInstitutional Animal Care and Use Committee. WT and FMR1 KO littermateswere produced by breeding heterozygous females with FMR1 KO males incongenic background of C57BL/6. The genotype of each animal wasconfirmed by PCR. For tissue collection, cortex were dissected, followedby protein isolation using Cellytic M (Sigma, St. Louis, Mo., USA) withproteinase inhibitors (Sigma, St. Louis, Mo., USA) according to themanufacturer's protocol.

Immunoblot Analysis

The proteins extracted from SH-SY5Y cells or cortex of FMR1 WT and KOmice were subjected to SDS-PAGE using NuPAGE Novex 4-20% Bis-Tris geland MOPS buffer (Invitrogen, Carlsbad, Calif., USA) according to themanufacturer's protocol. After electrophoresis, gels were electroblottedonto PVDF membranes (Millipore, Bedford, Mass., USA). Afterelectroblotting, membranes were blocked in SuperBlock blocking buffer(Pierce Biotechnology, Rockford, Ill., USA). Membranes were probed inthe blocking solution at 4° C. overnight with the following antibodies:FMRP (Chemicon, Temecula, Calif., USA), CYFIP1 (Upstate, Temecula,Calif., USA), JAKMIP1 (see, e.g. Steindler et al. (2004) J. Biol. Chem.,279, 43168-43177) or GAPDH. Membranes were washed 3× in PBS supplementedwith 0.05% Tween 20 (PBS-T) and incubated with the appropriatehorseradish peroxidase-conjugated secondary antibody in the blockingsolution for 1 hour at room temperature. Membranes were again washed 3×in PBS-T, developed with SuperSignal West Pico Chemiluminescent (PierceBiotechnology, Rockford, Ill., USA). Membranes were stripped by RestoreWestern Blot Stripping Buffer (Pierce Biotechnology, Rockford, Ill.,USA) and used for different antibodies.

Example 2 Genome-Wide Expression Profiling of Lymphoblastoid Cell LinesReveals Genes Dysregulated in Autism Spectrum Disorder

Autism spectrum disorder (ASD) is a heterogeneous condition and islikely to result from the combined effects of multiple, subtle geneticchanges interacting with environmental factors. We hypothesize thatthere are genes whose expression are deregulated in ASD. We believe thata subset of these genes can be identified through the whole genomeexpression profiling in lymphoblastoid cells from individuals withautism and control. Although lymphoblastoid cells are not neuronalcells, recent studies suggest that lymphoblastoid cells can be useful todetect biologically plausible correlations between candidate genes anddisease in various neuropsychiatric disorders. Our first study usinglymphoblastoid cells from ASD subjects with known genetic disordersshowed that genome-wide expression profiling of the lymphoblastoid celllines distinguishes different forms of ASD and reveals shared pathways.Here, we analyzed genome-wide expression profiling of lymphoblastoidcell lines from 15 male sib pairs discordant for idiopathic ASD. Weidentified genes dysregulated in common among the idiopathic ASD and ASDwith known genetic disorders. These results provide evidence that bloodderived lymphoblastoid cell gene expression is likely to be useful foridentifying susceptibility genes for ASD.

We previously reported that gene expression profiling of lymphoblastoidcell lines could identify different and shared pathways in cases ofautism spectrum disorder (ASD) with known genetic causes (see, e.g.Nishimura et al. 2007. Hum Mol Genet 16(14):1682-98). The analysisrevealed shared pathways between ASD with full mutation of FMR1(FMR1-FM) or maternal duplication of 15q11-q13 (dup15q), each of whichaccount for 1-2% of ASD cases in large series. Here, we analyzedgenome-wide expression profiling of lymphoblastoid cell lines from 15male sib pairs discordant for idiopathic ASD. We identified 95 genesdysregulated in common among the idiopathic ASD, ASD with FMR1-FM andASD with dup15q. We also identified 19 genes whose expression levelswere extremely different in one of the 15 affected males compared to themean of the 15 male sib pairs. We were able to confirm the differentialexpression of JAKMIP1, STEAP1, SLC16A6 and VIM between 39 male sib pairsdiscordant for ASD by quantitative PCR analysis. These results provideevidence that blood derived lymphoblastoid cell gene expression islikely to be useful for identifying susceptibility genes for ASD.

In this study, we analyzed the genome-wide expression profiles oflymphoblastoid cells from 15 male sib pairs discordant for idiopathicASD. ASD is heterogeneous condition that is likely to result from thecombined effects of multiple genetic factors (see, e.g. Abrahams et al.2008. Nat Rev Genet 9(5):341-55; Geschwind D H. 2008a. Nature454(7206):838-9; Geschwind D H. 2008b. Cell 135(3):391-5; and Geschwindet al. 2007. Curr Opin Neurobiol 17(1):103-11). Recent technologicaldevelopments, such as array-based comparative genomic hybridization(array-CGH), revealed strong association of de novo copy numbervariation (CNV) with ASD (see, e.g. Sebat et al. 2007. Science316(5823):445-9). However, each de novo CNV was individually rare in thepopulation of patients (see, e.g. Christian et al. 2008. Biol Psychiatry63(12):1111-7; Glessner et al. 2009. Nature; Marshall et al. 2008. Am JHum Genet 82(2):477-88; Sebat et al. 2007. Science 316(5823):445-9; andSzatmari et al. 20007. Nat Genet 39(3):319-28), suggesting that geneslocated within the CNV would be differentially expressed only in asubset of ASD. Although these genes may not be involved in ASD in thegeneral population, it is highly likely that they can provide essentialinformation with regard to biological pathways and genetic networksinvolved in the etiology of ASD. In this study, we focused our attentionon (i) genes that were dysregulated in common among idiopathic ASD, ASDwith FMR1-FM and ASD with dup15q and (ii) genes whose expression levelswere extremely different in a subset of the 15 affected males comparedto the mean of the 15 male sib pairs discordant for idiopathic ASD.Here, we demonstrate 92 genes dysregulated in common among the threedifferent forms of ASD and 19 genes whose expression levels wereextremely different (over 4 SD from the mean) in one of the 15 affectedmales. We were able to confirm the differential expression of januskinase and microtubule interacting protein 1 (JAKMIP1), sixtransmembrane epithelial antigen of the prostate 1 (STEAP1), solutecarrier family 16 member 6 (SLC16A6) and vimentin (VIM) between 39 malesib pairs discordant for idiopathic ASD by using quantitative PCRanalysis (qPCR). These results suggest that genome-wide expressionprofiling of lymphoblastoid cells is an efficient strategy to identifysusceptibility genes for ASD.

Materials and Methods

Individuals and Lymphoblastoid Cells Analyzed in this Study

We have analyzed individuals diagnosed with ASD using standard validatedmeasures, including the Autism Diagnostic Interview (ADI-R) (see, e.g.Lord et al. 1994. J Autism Dev Disord 24(5):659-85) and AutismDiagnostic Observation Schedule (ADOS) (see, e.g. Lord et al. 2001. Am JMed Genet 105(1):36-8), and controls (Table 3). All individuals weredrawn from AGRE (see, e.g. Geschwind et al. 2001. Am J Hum Genet69(2):463-6) (http://www.agre.org/). Lymphoblastoid cell lines (humanEpstein-Barr virus transformed lymphocytes) from these individuals wereavailable from AGRE. The lymphoblastoid cells of the subjects were grownas described previously (see, e.g. Nishimura et al. 2007. Hum Mol Genet16(14):1682-98).

Microarray Experiments

Microarray experiments were performed as described previously (see, e.g.Nishimura et al. 2007. Hum Mol Genet 16(14):1682-98). Scanner outputimage files from set 1 and 2 were normalized and filtered using FeatureExtraction Software v8.5 (Agilent, Santa Clara, Calif., USA).Normalization was performed so that overall intensity ratio of Cy5 toCy3 was equal to one.

Statistical Analysis of Microarray Data

To identify differentially expressed genes between groups, we analyzedthe probes that met following criteria in both Cy3 and Cy5 in at least13 of the 15 affected males. These criteria were i) signal was notsaturated, ii) signal was uniform, iii) signal-to-noise ratio was over2.6. The expression profile of these probes were subjected to EDGE (see,e.g. Leek et al. 2006. Bioinformatics 22(4):507-8) with 100 permutationsfor cross-validation. To identify differentially expressed genes (4 SDfrom the mean), we performed statistical analysis as described in (see,e.g. Coppola et al. 2008. Ann Neurol 64(1):92-6).

qPCR Analysis

One microgram of total RNA was used to make cDNA by iScript cDNASynthesis Kit (BioRad, Hercules, Calif., USA). The qPCR was performed asdescribed previously (see, e.g. Nishimura et al. 2007. Hum Mol Genet16(14):1682-98). The primers used in this study were JAKMIP1F;5′-GGGGAAGCATGTCGAAGAAA-3′ (SEQ ID NO: 1), JAKMIP1R;5′-GGCCTTGAGCTCCGAAATGT-3′3′ (SEQ ID NO: 2), STEAP1F; 5′-3′, STEAP1R;5′-3′, SLC16A6F; 5′-GGAGCCTTTGGGGGTTTATT-3′3′ (SEQ ID NO: 3), SLC16A6R;5′-CCATCCTCCATCAGGCACTT-3′3′ (SEQ ID NO: 4), VIMF;5′-AGCCGAAAACACCCTGCAATC-3′3′ (SEQ ID NO: 5), and VIMR;5′-CTGGATTTCCTCTTCGTGGAGTT-3′3′ (SEQ ID NO: 6).

Identification of Loci Associated with ASD

We used SLEP (see, e.g. Konneker et al. 2008. Am J Med Genet BNeuropsychiatr Genet 147B(6):671-5) to identify loci associated withASD. SLEP is a searchable archive of findings from psychiatric genetics.The database was queried by gene name in Table 1 using expansion of 5 Mbfor genome-wide linkage studies and 5 Kb for genome-wide associationstudies. We also used Autism Chromosomal Rearrangement Database toidentify de novo or overlapping CNVs involving genes identified in thisstudy.

Results

Microarray Analysis Identified 95 Genes Dysregulated in Idiopathic ASD,ASD with FMR1-FM and ASD with dup15q

We analyzed the whole-genome mRNA expression profiles in lymphoblastoidcells from 15 male sib pairs discordant for idiopathic ASD (Table 3) inthe Autism Genetic Resource Exchange (AGRE) (see, e.g. Geschwind et al.2001. Am J Hum Genet 69(2):463-6) using Agilent Whole Human GenomeArray. Overall, of 41,000 probes analyzed, 25497 probes, representing13893 genes, were expressed in the lymphoblastoid cells. To find thegenes that were differentially expressed between the 15 male sib pairsdiscordant for idiopathic ASD, the expression profile was subjected toExtraction of Differential Gene Expression (EDGE) (see, e.g. Leek et al.2006. Bioinformatics 22(4):507-8). EDGE uses newly developed statisticalmethodology including Optimal Discovery Procedure and shows substantialimprovements over five leading methodologies (see, e.g. Leek et al.2006. Bioinformatics 22(4):507-8). EDGE identified 579 probes belowP-value of 5% (FIG. 8). However, none of the probes had false discoveryrate (FDR) under 50%, suggesting genetic heterogeneity of these 15 malesib pairs. We previously analyzed genome-wide expression profile oflymphoblastoid cell lines from 6 males with ASD and FMR1-FM and 7 maleswith ASD and dup15q (see, e.g. Nishimura et al. 2007. Hum Mol Genet16(14):1682-98). These males comprise two homogeneous subsets of ASD. Wereanalyzed the expression profile using EDGE and identified 4033 and2196 probes dysregulated (FDR <5%) in ASD with FMR1-FM and ASD withdup15q, respectively (FIG. 8). 1065 probes, representing 752 genes, weredysregulated in both ASD with FMR1-FM and dup15q. Consistent with theprevious analysis (see, e.g. Nishimura et al. 2007. Hum Mol Genet16(14):1682-98), this overlap was highly significant (hypergeometricprobability, P=1.8×10⁻³¹⁷). We compared the 579 probes dysregulated inidiopathic ASD with probes dysregulated in ASD with FMR1-FM and/or ASDwith dup15q. As shown in FIG. 8, 208 and 171 probes were alsodysregulated in ASD with FMR1-FM and ASD with dup15q, respectively.Hypergeometric probability for these overlap were P=3.4×10⁻³³(idiopathic ASD and ASD with FMR1-FM) and P=3.0×10⁻⁴⁹ (idiopathic ASDand ASD with dup15q), suggesting these overlap were also significant.124 probes representing 95 genes were dysregulated in common among thethree different forms of all ASD (FIG. 8 and Table 1). Among the 95genes, 32 genes were located within the autism loci previouslyidentified by genetic analysis (Table 1) (see, e.g. Alarcon et al. 2002.Am J Hum Genet 70(1):60-71 Epub 2001 Dec. 6; Alarcon et al. 2005. MolPsychiatry 10(8):747-57; Allen-Brady et al. 2008. Mol Psychiatry;Auranen et al. 2002. Am J Hum Genet 71(4):777-90 Epub 2002 Aug. 21;Barrett et al. 1999. Am J Med Genet 88(6):609-15; Buxbaum et al. 2001.Am J Hum Genet 68(6):1514-20; Cantor et al. 2005. Am J Hum Genet76(6):1050-6; Duvall et al. 2007. Am J Psychiatry 164(4):656-62; IMGSAC.2001. Am J Hum Genet 69(3):570-81; Liu et al. 2001. Am J Hum Genet69(2):327-40 Epub 2001 Jul. 10; Marshall et al. 2008. Am J Hum Genet82(2):477-88; Schellenberg et al. 2006. Mol Psychiatry 11(11):1049-60,979; Sebat et al. 2007. Science 316(5823):445-9; Szatmari et al. 2007.Nat Genet 39(3):319-28; Trikalinos et al. 2006. Mol Psychiatry11(1):29-36; and Yonan et al. 2003. Am J Hum Genet 73(4):886-97 Epub2003 Sep. 17).

To provide functional classification of the 95 genes, we used IngenuityPathway Analysis (IPA). IPA identified four statistically significantnetworks, each containing at least 10 genes (Table 4 and FIG. 3).Principal functions associated with these networks were cellulardevelopment and cancer (Table 4).

Microarray Analysis Also Identified 19 Genes Whose Expression Levelswere Extremely Different in One of the 15 Affected Males

We then focused our attention on genes that were differentiallyexpressed (over 4 SD from the mean of 15 male sib pairs) in a subset ofthe 15 affected males. We identified 19 genes, each of which wasdifferentially expressed (1.7-15 fold change) in only one of the 15 malesib pairs (Table 2). Interestingly, 2 of the 19 genes, GABA A receptorα4 (GABRA4) and oligodendrocyte myelin glycoprotein (OMG) have beenreported to have significant association with ASD (see, e.g. Ma et al.2005. Am J Hum Genet 77(3):377-88; and Martin et al. 2007b. Neurosci Res59(4):426-30). GABRA4, nicotinate phosphoribosyltransferase domaincontaining 1 (NAPRT1) and vimentin (VIM) were also located within CNVsthat occurred de novo or overlapped in ASD (see, e.g. Kakinuma et al.2007. Am J Med Genet B Neuropsychiatr Genet; and Weidmer-Mikhail et al.1998. J Intellect Disabil Res 42 (Pt 1):8-12).

qPCR Confirmed the Differential Expression of JAKMIP1, STEAP1, SLC16A6and VIM Between 39 Male Sib Pairs Discordant for Idiopathic ASD

To validate the differential expression identified by microarrayanalysis using independent methods, we performed quantitative PCRanalysis (qPCR) of JAKMIP1, STEAP1, SLC16A6 and VIM using lymphoblastoidcell lines from 39 male sib pairs discordant for idiopathic ASD (Table3). The 39 male sib pairs included the 15 male sib pairs analyzed in themicroarray study. JAKMIP1 was selected because we previouslydemonstrated that the expression of JAKMIP1 was significantly differentbetween 27 male sib pairs discordant for idiopathic ASD (see, e.g.Nishimura et al. 2007. Hum Mol Genet 16(14):1682-98). STEAP1, SLC16A6and VIM were selected because the microarray analysis identified thedysregulation of these genes by multiple probes (Table 1 and 2). Toattempt to reduce the heterogeneity of idiopathic ASD, we used an IQsurrogate based on Raven's Progressive Matrices. The 39 affected maleshad an IQ score of more than 70 (Table 3). As shown in FIG. 9, qPCRconfirmed the dysregulation of JAKMIP1, STEAP1, and SLC16A6 as expectedby the microarray analysis. qPCR also confirmed the reduction of VIM inone affected male as expected in the microarray analysis (6-foldreduction). The expression of VIM was significantly different betweenthe 39 male sib pairs discordant for idiopathic ASD.

In this study, we performed global mRNA expression profiling oflymphoblastoid cell line from 15 male sib pairs discordant foridiopathic ASD. We focused our attention on (i) genes that weredysregulated in common among idiopathic ASD, ASD with FMR1-FM and ASDwith dup15q and (ii) genes whose expression were extremely different (4standard deviation from the mean of the 15 male sib pairs) in a subsetof the 15 affected males.

Genes Dysregulated in Idiopathic ASD, ASD with FMR1-FM and ASD withdup15q

We first tried to identify genes that were differentially expressedbetween the 15 male sib pairs discordant for idiopathic ASD. 579 probeswere differentially expressed between the 15 male sib pairs. However,none of the 579 probes had FDR fewer than 50%, suggesting geneticheterogeneity of the 15 male sib pairs. It may also reflect that ASD canresult from many different genetic defects. Given the complexity of ASD,the study of ASD associated with Mendelian single gene disorders orknown chromosomal etiologies provides an important perspective (see,e.g. Belmonte et al 2006. Nat Neurosci 9(10):1221-5; and Geschwind etal. 2007. Curr Opin Neurobiol 17(1):103-11). Previously, we analyzedgenome-wide expression profiles of lymphoblastoid cells from ASD withFMR1-FM or dup15q and identified shared pathways between these ASD (see,e.g. Nishimura et al. 2007. Hum Mol Genet 16(14):1682-98). Wehypothesized that there might be genes dysregulated in common amongidiopathic ASD, ASD with FMR1-FM and ASD with dup15q. Comparing theexpression profiles, we identified 95 genes that were dysregulated incommon among the idiopathic ASD, ASD with FMR1-FM and ASD with dup15q.qPCR analysis confirmed the differential expression of JAKMIP1, STEAP1and SLC16A6 between 36 male sib pairs discordant for idiopathic ASD.

JAKMIP1 is associated with Janus kinases (see, e.g. Steindler et al.2004. J Biol Chem 279(41):43168-77), microtubules (see, e.g. Steindleret al. 2004. J Biol Chem 279(41):43168-77) and GABRB receptors (see,e.g. Couve et al. 2004. J Biol Chem 279(14):13934-43). Because GABRBreceptors could interact with the metabotropic receptor 1 (mGluR1) andincrease the glutamate sensitivity of mGluR1 (Tabata, et al., 2004),JAKMIP1 might affect mGluR1 signaling through GABRB receptors. GABAergicand glutamergic signaling have been reported to be dysregulated in ASD(Belmonte and Bourgeron, 2006). Two individuals with ASD were reportedto have CNVs containing JAKMIP1 (see, e.g. Sebat et al. 2007. Science316(5823):445-9; and Szatmari et al. 2007. Nat Genet 39(3):319-28),suggesting dysregulation of JAKMIP1 may be involved in the etiology ofASD.

Although the function of STEAP1 is currently unknown, other members ofSTEAP family are not only ferrireductase but also cupric reductase thatstimulate cellular uptake of both iron and copper (see, e.g. Ohgami etal. 2006. Blood 108(4):1388-94). Epitope-tagged STEAP1 expressed inHEK293-T cells partially colocalized with transferrin (Tf) and Tfreceptor, suggesting STEAP1 might modulate iron-transport through Tf/Tfreceptor (see, e.g. Ohgami et al. 2006. Blood 108(4):1388-94). It isimportant to note that levels of Tf and ceruloplamsin (copper-bindingprotein) in the serum were significantly reduced in children with ASD ascompared to their developmentally normal siblings (see, e.g. Chauhan etal. 2004. Life Sci 75(21):2539-49). These findings suggest that STEAP1may be related to the pathophysiology of ASD. It was shown that a SNP(rs4015735) could be a regulatory variant in expression of STEAP1 andoptimal for investigations in case-control studies (see, e.g. Ge et al.2005. Genome Res 15(11):1584-91). Genetic association studies betweenthe STEAP1 polymorphism and high-functioning ASD might be worth doing.

SLC16A6 is a putative monocarboxylate transporter although the functionhas not been characterized. The expression of SLC16A6 was reduced byIL-13 in human monocyte (see, e.g. Scotton et al. 2005. J Immunol174(2):834-45). Elevation of IL-13 levels was reported in children withASD (see, e.g. Molloy et al. 2006. J Neuroimmunol 172(1-2):198-205).Interestingly, we identified IL-13 receptor α1 (IL13RA1) as anup-regulated gene in idiopathic ASD, ASD with FMR1-FM, ASD with dup15q(Table 1). It is also important to note that SLC16A6 is located withinregions that were identified as an autism locus by genetic analyses(see, e.g. Alarcon et al. 2005. Mol Psychiatry 10(8):747-57). Thesefindings suggest that IL-13 signaling involving SLC16A6 might bedysregulated in ASD.

Genes Extremely Dysregulated in One of the 15 Affected Males

It has been shown that individuals with ASD carry chromosomalabnormality at a greater frequency than the general population (see,e.g. Christian et al. 2008. Biol Psychiatry 63(12):1111-7; Glessner etal. 2009. Nature; Jacquemont et al. 2006. J Med Genet 43(11):843-9;Marshall et al. 2008. Am J Hum Genet 82(2):477-88; Sebat et al. 2007.Science 316(5823):445-9; Szatmari et al. 2007. Nat Genet 39(3):319-28;Veenstra-Vanderweele et al. 2004. Annu Rev Genomics Hum Genet 5:379-405;and Vorstman et al. 2006. Mol Psychiatry 11(1):1, 18-28). Many studiesfind rates of detected abnormalities in 5-10% of affected individuals(see, e.g. Veenstra-Vanderweele et al. 2004. Annu Rev Genomics Hum Genet5:379-405; and Vorstman et al. 2006. Mol Psychiatry 11(1):1, 18-28).These abnormalities include unbalanced translocation, inversions, rings,and interstitial or terminal deletions and duplications (see, e.g.Vorstman et al. 2006. Mol Psychiatry 11(1):1, 18-28). The most frequentfinding in ASD is dup15q (see, e.g. Szatmari et al. 2007. Nat Genet39(3):319-28; Veenstra-Vanderweele et al. 2004. Annu Rev Genomics HumGenet 5:379-405; and Vorstman et al. 2006. Mol Psychiatry 11(1):1,18-28). Deletions of 2q37 and 22q13.3 have also been reported more thanone occasion (see, e.g. Vorstman et al. 2006. Mol Psychiatry 11(1):1,18-28). These chromosomal abnormalities can influence gene dosage andexpression (see, e.g. Feuk et al. 2006. Hum Mol Genet 15 Spec No1:R57-66). However, many chromosomal abnormalities are individually rarein the population with ASD. We hypothesized that genes whose expressionlevels were extremely different from the means of all individuals mightbe related to chromosomal abnormalities associated with ASD and that thedifferential expression might be detected in only one or a few affectedmales in the 15 male sib pairs. In this study, we identified 19 geneswhose expression levels were extremely different in one of the 15affected males. These genes included VIM, OMG, and GABRA4.

It has been reported that VIM is regulated by FMR1 (see, e.g. Miyashiroet al. 2003. Neuron 37(3):417-31; and Nishimura et al. 2007. Hum MolGenet 16(14):1682-98) and TSC1/2 (see, e.g. Hengstschlager et al. 2004.Cancer Lett 210(2):219-26) that are causative genes for Fragile Xsyndrome and Tuberous sclerosis, respectively. Clinical phenotypes ofthese syndromes include ASD, suggesting VIM may be involved in theetiology of ASD. qPCR analysis confirmed the large reduction of VIM inone of the 15 affected male as expected in the microarray analysis. Themechanism of the 6-fold reduction of VIM is unknown. VIM has also beeninvolved in CNVs that occurred in ASD (Sahoo et al. ASHG2006, #822).Interestingly, qPCR analysis also demonstrated that the expression ofVIM was significantly different between the 39 male sib pairs discordantfor idiopathic ASD.

Both OMG and GABRA4 have been reported to have significant associationwith ASD (see, e.g. Ma et al. 2005. Am J Hum Genet 77(3):377-88; andMartin et al. 2007b. Neurosci Res 59(4):426-30). An association betweena subgroup of French patients with ASD and an allele of a non-synonymousSNP (rs11080149) in OMG were reported (see, e.g. Martin et al. 2007b.Neurosci Res 59(4):426-30). The SNP consisted in a G to A transition atposition 62 from the start codon, changing a glycine to an asparticacid. The amino acid change may modulate the precise localization ormaturation of OMG (see, e.g. Martin et al. 2007b. Neurosci Res59(4):426-30). The mechanism and meaning of the over-expression of OMGin ASD remain to be studied. rs1912960 in GABRA4 was also reported tohave significant allelic and genotypic association with ASD (see, e.g.Ma et al. 2005. Am J Hum Genet 77(3):377-88). Recently, an autism casewith duplication of GABRA4 gene was reported (see, e.g. Kakinuma et al.2007. Am J Med Genet B Neuropsychiatr Genet). Dysregulation of GABRA4may be relevant to etiology of ASD.

Recently, Zhang et al surveyed expression profile of X-linked genes inlymphoblastoid cells from 43 males with X-linked mental retardation(XLMR) (see, e.g. Zhang et al. 2007. Genome Res 17(5):641-8). Theyidentified 15 candidate genes including proteolipid protein 2 (PLP2).These genes were dysregulated only in one or two patients among the 47males with XLMR. However, they identified a functional PLP2 promoterpolymorphism enriched in patients with XLMR using large cohort (see,e.g. Zhang et al. 2007. Genome Res 17(5):641-8). These findings suggestthat there might be regulatory polymorphisms in the 19 genes enriched inpatients with ASD.

CNV may also be the cause of the dysregulation of these 19 genesidentified in this study. It has been shown that CNV can alter mRNAexpression (see, e.g. Durand et al. 2007. Nat Genet 39(1):25-7; Jeffrieset al. 2005. Am J Med Genet A 137(2):139-47; Nishimura et al. 2007. HumMol Genet 16(14):1682-98; and Stranger et al. 2007. Science315(5813):848-53) and de novo CNV strongly associated with ASD (see,e.g. Sebat et al. 2007. Science 316(5823):445-9). Further study isneeded to analyze CNV of the 19 genes in individuals identified in thisstudy and in large cohort.

In conclusion, this study provides evidences that genome-wide expressionprofiling of lymphoblastoid cells is useful to identify susceptibilitygenes for ASD.

Tables

TABLE 1 Genes dysregulated in common among idiopathic ASD, ASD withFMR1-FM and ASD with dup15q Probe Symbol Gene Name Gene Name LocusIdio/C FM/C Dup/C A_24_P342591 RERE arginine-glutamic arginine-glutamic−0.06 −0.16 −0.14 acid dipeptide acid dipeptide (RE) repeats (RE)repeats A_23_P257365 GFI1 growth factor growth factor −0.12 −0.17 −0.22independent 1 independent 1 A_32_P150300 LOC100131646 LOC100131646LOC100131646 −0.06 −0.16 −0.16 A_24_P128163 ADAMTS4 ADAM ADAM −0.07−0.12 −0.16 metallopeptidase metallopeptidase with thrombospondin withthrombospondin type 1 motif, 4 type 1 motif, 4 A_23_P114929 BRP44 brainprotein 44 brain protein 44 −0.07 −0.17 −0.20 A_24_P234196 RRM2ribonucleotide ribonucleotide −0.09 −0.19 −0.28 reductase M2 reductaseM2 polypeptide polypeptide A_23_P120153 RNF149 ring finger ring finger−0.06 −0.20 −0.15 protein 149 protein 149 A_23_P209995 IL1RN interleukin1 interleukin 1 0.05 0.14 0.06 receptor antagonist receptor antagonistA_23_P360079 NAP5 Nck-associated Nck-associated −0.19 −0.29 −0.28protein 5 protein 5 A_24_P484965 LOC730124 LOC730124 LOC730124 0.04 0.090.07 A_24_P208452 BBS5 Bardet-Biedl Bardet-Biedl 0.07 0.19 0.21 Buxbaumsyndrome 5 syndrome 5 2001 A_23_P17130 MGC13057 −0.09 −0.25 −0.38A_23_P131676 CXCR7 chemokine (C-X-C chemokine (C-X-C 0.16 0.43 0.66Sebat motif) receptor 7 motif) receptor 7 2007 A_23_P259362 NPCDR1nasopharyngeal nasopharyngeal 0.02 0.06 0.06 carcinoma, down- carcinoma,down- regulated 1 regulated 1 A_23_P253250 GCET2 germinal centergerminal center 0.13 0.61 0.51 Allen- expressed expressed Bradytranscript 2 transcript 2 2008 A_24_P182947 GCET2 germinal centergerminal center 0.14 0.59 0.61 Allen- expressed expressed Bradytranscript 2 transcript 2 2008 A_23_P253317 GPR171 G protein-coupled Gprotein-coupled −0.13 −0.24 −0.35 Alarcon receptor 171 receptor 171 2005A_23_P351215 SKIL SKI-like SKI-like −0.10 −0.24 −0.15 Alarcon 2005,Allen- Brady 2008 A_23_P58036 MCCC1 methylcrotonoyl- methylcrotonoyl-0.05 0.08 0.14 Allen- Coenzyme A Coenzyme A Brady carboxylase 1carboxylase 1 2008 (alpha) (alpha) A_23_P144274 JAKMIP1 janus kinase andjanus kinase and 0.29 1.00 0.61 microtubule microtubule interactingprotein 1 interacting protein 1 A_23_P18465 RFC1 replication factor Creplication factor C −0.05 −0.10 −0.09 (activator 1) 1, (activator 1) 1,145 kDa 145 kDa A_32_P165477 SLC7A11 solute carrier family solutecarrier family −0.08 −0.23 −0.17 Schellenberg 7 member 11 7 member 112006 A_23_P121885 ROPN1L ropporin 1-like ropporin 1-like 0.07 0.12 0.10Marshall 2008 A_32_P154342 SLCO4C1 solute carrier solute carrier −0.14−0.33 −0.53 organic anion organic anion transporter family, transporterfamily, member 4C1 member 4C1 A_24_P409042 CDC42SE2 CDC42 small CDC42small −0.07 −0.13 −0.09 effector 2 effector 2 A_23_P310972 PCDHGB1protocadherin protocadherin 0.03 0.05 0.05 gamma subfamily gammasubfamily B, 1 B, 1 A_23_P7503 TIMD4 T-cell T-cell −0.29 −0.53 −0.57immunoglobulin immunoglobulin and mucin domain and mucin domaincontaining 4 containing 4 A_32_P356316 HLA-DOA major major 0.10 0.280.25 histocompatibility histocompatibility complex, class II, complex,class II, DO alpha DO alpha A_24_P288836 HLA-DPB2 major major 0.09 0.240.21 histocompatibility histocompatibility complex, class II, complex,class II, DP beta 2 DP beta 2 A_23_P42353 ETV7 ets variant gene 7 etsvariant gene 7 0.10 0.26 0.39 A_24_P334640 PAQR8 progestin and progestinand 0.07 0.21 0.18 adipoQ receptor adipoQ receptor family member VIIIfamily member VIII A_23_P255952 MYO6 myosin VI myosin VI −0.13 −0.40−0.26 A_23_P350451 PRDM1 PR domain PR domain −0.15 −0.34 −0.17containing 1, with containing 1, with ZNF domain ZNF domain A_23_P93442SASH1 SAM and SH3 SAM and SH3 −0.17 −0.62 −0.73 domain containing 1domain containing 1 A_24_P135841 LRP11 low density low density −0.13−0.36 −0.30 lipoprotein lipoprotein receptor-related receptor-relatedprotein 11 protein 11 A_23_P111593 PSCD3 pleckstrin pleckstrin 0.09 0.210.34 homology, Sec7 homology, Sec7 and coiled-coil and coiled-coildomains 3 domains 3 A_23_P252145 C1GALT1 core 1 synthase core 1 synthase−0.07 −0.21 −0.22 A_23_P31453 STEAP1 six transmembrane six transmembrane−0.26 −0.73 −0.46 Barrett epithelial antigen epithelial antigen 1999 ofthe prostate 1 of the prostate 1 A_24_P406334 STEAP1 six transmembranesix transmembrane −0.21 −0.58 −0.38 Barrett epithelial antigenepithelial antigen 1999 of the prostate 1 of the prostate 1 A_32_P69149STEAP1 six transmembrane six transmembrane −0.24 −0.65 −0.46 Barrettepithelial antigen epithelial antigen 1999 of the prostate 1 of theprostate 1 A_23_P95130 SLC37A3 solute carrier solute carrier 0.08 0.170.12 Alarcon family 37 member 3 family 37 member 3 2002, Trikalinos2005, Arkin 2008 A_24_P274831 GIMAP7 GTPase, IMAP GTPase, IMAP 0.27 0.530.48 Alarcon family member 7 family member 7 2002, Trikalinos 2005,Arkin 2008 A_23_P427023 GIMAP1 GTPase, IMAP GTPase, IMAP 0.20 0.42 0.43Alarcon family member 1 family member 1 2002, Trikalinos 2005, Arkin2008 A_24_P92624 GIMAP5 GTPase, IMAP GTPase, IMAP 0.11 0.20 0.29 Alarconfamily member 5 family member 5 2002, Trikalinos 2005, Arkin 2008A_23_P31810 CEBPD CCAAT/enhancer CCAAT/enhancer −0.17 −0.38 −0.39binding protein binding protein (C/EBP), delta (C/EBP), deltaA_23_P112135 TRAM1 translocation translocation −0.09 −0.24 −0.21associated associated membrane protein 1 membrane protein 1 A_23_P146990WWP1 WW domain WW domain −0.04 −0.07 −0.07 containing E3 containing E3ubiquitin protein ubiquitin protein ligase 1 ligase 1 A_32_P176675FAM92A1 family with family with 0.09 0.13 0.20 sequence similaritysequence similarity 92, member A1 92, member A1 A_23_P168951 ZHX2 zincfingers and zinc fingers and −0.08 −0.15 −0.10 Yonan homeoboxes 2homeoboxes 2 2003 A_23_P111978 KCNK9 potassium channel, potassiumchannel, −0.06 −0.17 −0.14 subfamily K, subfamily K, member 9 member 9A_23_P94552 TMEM2 transmembrane transmembrane −0.20 −0.59 −0.56 protein2 protein 2 A_24_P84419 VAV2 vav 2 oncogene vav 2 oncogene 0.04 0.080.09 IMGSAC 2001, Auranen 2002 A_23_P217120 EHMT1 euchromaticeuchromatic −0.05 −0.16 −0.19 IMGSAC histone-lysine N- histone-lysine N-2001, methyltransferase 1 methyltransferase 1 Auranen 2002 A_24_P374834OTUD1 OTU domain OTU domain −0.13 −0.23 −0.30 containing 1 containing 1A_32_P60459 OTUD1 OTU domain OTU domain −0.13 −0.29 −0.24 containing 1containing 1 A_23_P52207 BAMBI BMP and activin BMP and activin −0.38−0.67 −0.51 membrane-bound membrane-bound inhibitor homolog inhibitorhomolog A_23_P86623 ENTPD7 ectonucleoside ectonucleoside −0.04 −0.13−0.13 triphosphate triphosphate diphosphohydrolase 7 diphosphohydrolase7 A_23_P158570 ACADSB acyl-Coenzyme A acyl-Coenzyme A 0.04 0.18 0.15dehydrogenase, dehydrogenase, short/branched short/branched chain chainA_24_P189516 ACADSB acyl-Coenzyme A acyl-Coenzyme A 0.06 0.12 0.10dehydrogenase, dehydrogenase, short/branched short/branched chain chainA_32_P215002 CD44 CD44 molecule CD44 molecule 0.09 0.11 0.19 Trikalinos2005, Szatmari 2007, Duvall 2007 A_24_P13083 TSPAN18 tetraspanin 18tetraspanin 18 0.06 0.10 0.09 Duvall 2007 A_23_P2143 SPCS2 signalpeptidase signal peptidase −0.06 −0.14 −0.09 Duvall complex subunit 2complex subunit 2 2007 homolog homolog A_23_P150768 SLCO2B1 solutecarrier solute carrier −0.16 −0.32 −0.40 Duvall organic anion organicanion 2007 transporter family, transporter family, member 2B1 member 2B1A_23_P1775 DPAGT1 dolichyl-phosphate dolichyl-phosphate −0.03 −0.06−0.08 Duvall N-acetylglucosamine- N-acetylglucosamine 2007phosphotransferase 1 phosphotransferase 1 A_24_P241183 CLEC2D C-typelectin C-type lectin −0.14 −0.28 −0.36 domain family 2, domain family 2,member D member D A_24_P52921 BCAT1 branched chain branched chain 0.100.19 0.24 aminotransferase aminotransferase 1, cytosolic 1, cytosolicA_23_P306507 KRAS v-Ki-ras2 Kirsten v-Ki-ras2 Kirsten −0.13 −0.22 −0.20rat sarcoma viral rat sarcoma viral oncogene homolog oncogene homologA_32_P2452 TMTC1 transmembrane and transmembrane and −0.22 −0.68 −0.52tetratricopeptide tetratricopeptide repeat containing 1 repeatcontaining 1 A_24_P98914 PFKM phosphofructokinase, phosphofructokinase,0.03 0.10 0.08 muscle muscle A_24_P832426 B3GALTL beta 1,3- beta 1,3-−0.13 −0.25 −0.18 Barrett galactosyltransferase- galactosyltransferase-1999 like like A_23_P428738 ANG angiogenin, angiogenin, −0.13 −0.18−0.23 ribonuclease, ribonuclease, RNase A family, 5 RNase A family, 5A_23_P151586 TM9SF1 transmembrane 9 transmembrane 9 −0.04 −0.15 −0.10superfamily superfamily member 1 member 1 A_23_P65518 DACT1 dapper,antagonist dapper, antagonist −0.26 −0.42 −0.50 of beta-catenin, ofbeta-catenin, homolog 1 homolog 1 A_23_P348936 CTAGE5 CTAGE family,CTAGE family, −0.06 −0.11 −0.13 member 5 member 5 A_23_P88381 NUMB numbhomolog numb homolog −0.07 −0.12 −0.10 A_23_P163306 CGNL1 cingulin-like1 cingulin-like 1 −0.13 −0.31 −0.26 A_23_P65779 STRA6 stimulated bystimulated by 0.04 0.05 0.11 Szatmari retinoic acid gene retinoic acidgene 2007, 6 homolog 6 homolog Marshall 2008 A_23_P129128 TARSL2threonyl-tRNA threonyl-tRNA −0.11 −0.19 −0.11 synthetase-like 2synthetase-like 2 A_23_P129556 IL4R interleukin 4 interleukin 4 0.100.19 0.20 receptor receptor A_24_P227927 IL21R interleukin 21interleukin 21 0.06 0.21 0.17 receptor receptor A_23_P206822 XPO6exportin 6 exportin 6 0.04 0.07 0.07 A_23_P3681 NETO2 neuropilin (NRP)neuropilin (NRP) −0.06 −0.14 −0.12 and tolloid (TLL)- and tolloid (TLL)-like 2 like 2 A_23_P14946 MBTPS1 membrane-bound membrane-bound −0.06−0.15 −0.13 transcription factor transcription factor peptidase, site 1peptidase, site 1 A_23_P14948 MBTPS1 membrane-bound membrane-bound −0.05−0.18 −0.15 transcription factor transcription factor peptidase, site 1peptidase, site 1 A_23_P4294 ZNF232 zinc finger zinc finger 0.05 0.140.08 Duvall protein 232 protein 232 2007 A_24_P188218 MYL4 myosin, lightchain myosin, light chain −0.13 −0.26 −0.23 Cantor 4, alkali; atrial, 4,alkali; atrial, 2005, embryonic embryonic Duvall 2007 A_23_P89455SLC35B1 solute carrier family solute carrier family −0.04 −0.10 −0.10Cantor 35, member B1 35, member B1 2005, Duvall 2007 A_32_P217346 APPBP2amyloid beta amyloid beta 0.04 0.05 0.15 Alarcon precursor proteinprecursor protein 2005, binding protein 2 binding protein 2 Cantor 2005,Duvall 2007 A_23_P152791 SLC16A6 solute carrier family solute carrierfamily −0.06 −0.22 −0.26 Alarcon 16, member 6 16, member 6 2005A_24_P731648 SLC16A6 solute carrier family solute carrier family −0.07−0.24 −0.23 Alarcon 16, member 6 16, member 6 2005 A_23_P412577 ANKRD29ankyrin repeat ankyrin repeat −0.12 −0.25 −0.32 domain 29 domain 29A_23_P66948 FAM59A family with family with −0.20 −0.49 −0.43 sequencesimilarity sequence similarity 59, member A 59, member A A_23_P433063ATCAY ataxia, cerebellar, ataxia, cerebellar, 0.03 0.05 0.06Schellenberg Cayman type Cayman type 2006 A_23_P50426 KANK2 KN motif andKN motif and −0.09 −0.23 −0.21 Liu ankyrin repeat ankyrin repeat 2001domains 2 domains 2 A_24_P93887 MED29 mediator complex mediator complex0.03 0.09 0.07 Liu subunit 29 subunit 29 2001 A_23_P166100 TXNDC13thioredoxin domain thioredoxin domain −0.09 −0.21 −0.22 containing 13containing 13 A_23_P17316 NKAIN4 Na+/K+ Na+/K+ 0.05 0.05 0.11 Marshalltransporting transporting 2008 ATPase interacting 4 ATPase interacting 4A_24_P339869 ZNF295 zinc finger zinc finger 0.05 0.11 0.09 protein 295protein 295 A_24_P267686 LOC729314 LOC729314 LOC729314 0.04 0.05 0.10A_24_P387514 LRP5L low density low density 0.05 0.16 0.08 lipoproteinlipoprotein receptor-related receptor-related protein 5-like protein5-like A_24_P945283 DLG3 discs, large discs, large −0.16 −0.30 −0.47homolog 3 homolog 3 A_23_P137196 IL13RA1 interleukin 13 interleukin 130.11 0.20 0.26 receptor, alpha 1 receptor, alpha 1 A_24_P280113 IL13RA1interleukin 13 interleukin 13 0.29 0.51 0.61 receptor, alpha 1 receptor,alpha 1 A_23_P213085 unknown −0.07 −0.16 −0.18 A_24_P152775 unknown 0.030.05 0.10 A_24_P221285 unknown −0.09 −0.19 −0.30 A_24_P238118 unknown0.03 0.07 0.08 A_24_P384979 unknown 0.05 0.10 0.17 A_24_P479510 unknown−0.13 −0.21 −0.26 A_24_P493100 unknown −0.04 −0.11 −0.15 A_24_P521662unknown 0.04 0.09 0.11 A_24_P63397 unknown 0.03 0.13 0.10 A_24_P68079unknown −0.09 −0.27 −0.18 A_24_P925310 unknown 0.05 0.07 0.11A_32_P102383 unknown 0.04 0.07 0.21 A_32_P103815 unknown −0.09 −0.15−0.18 A_32_P137826 unknown −0.20 −0.32 −0.45 A_32_P163894 unknown 0.050.15 0.21 A_32_P232682 unknown −0.10 −0.16 −0.17 A_32_P34696 unknown0.06 0.12 0.11 A_32_P45309 unknown 0.04 0.14 0.13 A_32_P69333 unknown−0.12 −0.39 −0.27 A_32_P72758 unknown −0.14 −0.42 −0.58 A_32_P9924unknown 0.04 0.09 0.06 ^(a)idio/CNT was log₂ (mean value of control (N =15)/mean value of idiopathic ASD (N = 15)). ^(b)FM/CNT was log₂ (meanvalue of ASD with FMR1FM (N = 6)/mean value of idiopathic ASD (N = 15)).^(c)dup/CNT was log₂ (mean value of ASD with dup15q (N = 7)/mean valueof idiopathic ASD (N = 15)). ^(d)Autism loci identified by other geneticstudies were shown with references.

TABLE 2 Genes extremely dysregulated in one of the 15 affected malesProbe Symbol Gene Name Locus Change Individual Reference A_23_P149529TACSTD2 tumor-associated calcium 1p32-p31 2.1 AU1038302 signaltransducer 2 A_24_P222147 C1orf131 chromosome 1 open reading 1q42.2 1.0AU055105 frame 131 A_24_P169234 ZAP70 zeta-chain associated 2q12 1.2AU055105 protein kinase 70 kDa A_23_P79518 IL1B interleukin 1, beta 2q142.4 AU0943301 A_32_P204137 GABRA4 GABA A receptor, alpha 4 4p12 2.2AU016803 Kakinuma 2008 A_23_P122443 HIST1H1C histone 1, H1c 6p21.3 −2.4AU0943301 A_24_P280628 VPS13B vacuolar protein sorting 8q22.2 −1.1AU1215304 13B A_23_P258312 NAPRT1 nicotinate 8q24.3 −3.1 AU0943301Weidmer- phosphoribosyltransferase Mikhail domain containing 1 1998A_32_P395992 DEC1 deleted in esophageal 9q32 2.3 AU0943301 cancer 1A_23_P161190 VIM vimentin 10p13 −2.6 AU0943301 Sahoo et. al ASHG 2006A_23_P161194 VIM vimentin 10p13 −2.6 AU0943301 Newman et al ASHG 2006A_23_P151046 KLRC1 killer cell lectin-like 12p13 2.1 AU081205 receptorsubfamily C, member 1 A_24_P409126 FNDC3A fibronectin type III domain13q14.2 1.8 AU055105 containing 3A A_23_P48530 INSM2insulinoma-associated 2 14q13.2 1.0 AU055105 A_23_P65629 KCNK10potassium channel, 14q31 0.8 AU055105 subfamily K, member 10 A_23_P55286OMG oligodendrocyte myelin 17q11.2 2.0 AU016803 glycoproteinA_23_P119353 RASIP1 Ras interacting protein 1 19q13.33 3.9 AU1165302A_23_P21120 MED14 mediator complex subunit Xp11.4-p11.2 1.1 AU016803 14A_23_P432352 CXorf61 chromosome X open Xq23 1.7 AU016803 reading frame61 A_23_P73677 RHOXF2 Rhox homeobox family, Xq24 1.4 AU1157301 member 2A_23_P352494 RHOXF2 Rhox homeobox family, Xq24 1.4 AU1157301 member 2^(a)Change was the value of log₂ (intensity of the proband/mean value ofthe 15 male sib pairs).

REFERENCES CITED IN TABLES 1 AND 2

Alarcon et al. 2002. Am J Hum Genet 70(1):60-71 Epub 2001 Dec. 6.

Alarcon et al. 2005. Mol Psychiatry 10(8):747-57.

Allen-Brady et al. Feb. 19, 2008. Mol Psychiatry.

Auranen et al. 2002. Am J Hum Genet 71(4):777-90 Epub 2002 Aug. 21.

Barrett et al. 1999. Am J Med Genet 88(6):609-15.

Buxbaum et al. 2001. Am J Hum Genet 68(6):1514-20.

Cantor et al. 2005. Am J Hum Genet 76(6):1050-6.

Duvall et al. 2007. Am J Psychiatry 164(4):656-62.

Kakinuma et al. 2007. Am J Med Genet B Neuropsychiatr Genet.

Liu et al. 2001. Am J Hum Genet 69(2):327-40 Epub 2001 Jul. 10.

Marshall et al. 2008. Am J Hum Genet 82(2):477-88.

Schellenberg et al. 2006. Mol Psychiatry 11(11):1049-60, 979.

Sebat et al. 2007. Science 316(5823):445-9.

Szatmari et al. 2007. Nat Genet 39(3):319-28.

Trikalinos et al. 2006. Mol Psychiatry 11(1):29-36.

Weidmer-Mikhail et al. 1998. J Intellect Disabil Res 42 (Pt 1):8-12.

Yonan et al. 2003. Am J Hum Genet 73(4):886-97 Epub 2003 Sep. 17.

TABLE 3 Individuals analyzed in this study Individual Code family IDgroup ADIR ADOS Raven IQ analysis AU016703 AU0167 CNT microarray, qPCRAU016704 AU0167 idiopathic BroadSpectrum Spectrum 100 microarray, ASDqPCR AU016803 AU0168 idiopathic Autism Autism 94 microarray, ASD qPCRAU016804 AU0168 CNT microarray, qPCR AU055103 AU0551 CNT microarray,qPCR AU055105 AU0551 idiopathic NQA Spectrum 108 microarray, ASD qPCRAU060003 AU0600 CNT microarray, qPCR AU060004 AU0600 idiopathic Autism100 microarray, ASD qPCR AU081205 AU0812 idiopathic Autism not Spectrum128 microarray, ASD or Autism qPCR AU081206 AU0812 CNT microarray, qPCRAU0943301 AU0943 idiopathic Autism Spectrum 110 microarray, ASD qPCRAU0943303 AU0943 CNT microarray, qPCR AU0995302 AU0995 CNT microarray,qPCR AU0995303 AU0995 idiopathic Autism Spectrum 105 microarray, ASDqPCR AU1038302 AU1038 idiopathic Autism Spectrum 95 microarray, ASD qPCRAU1038304 AU1038 CNT microarray, qPCR AU1086301 AU1086 CNT microarray,qPCR AU1086303 AU1086 idiopathic Autism Autism 110 microarray, ASD qPCRAU1157301 AU1157 idiopathic Autism Autism 119 microarray, ASD qPCRAU1157302 AU1157 CNT microarray, qPCR AU1165302 AU1165 idiopathic AutismAutism 110 microarray, ASD qPCR AU1165303 AU1165 CNT microarray, qPCRAU1165304 AU1165 idiopathic Autism Autism 114 microarray, ASD qPCRAU1165305 AU1165 CNT microarray, qPCR AU1215303 AU1215 CNT microarray,qPCR AU1215304 AU1215 idiopathic Autism Spectrum 119 microarray, ASDqPCR AU1327302 AU1327 idiopathic Autism Autism 104 microarray, ASD qPCRAU1327303 AU1327 CNT microarray, qPCR AU1348302 AU1348 CNT microarray,qPCR AU1348303 AU1348 idiopathic Autism Autism 107 microarray, ASD qPCRAU0081302 AU0081 CNT qPCR AU0081303 AU0081 idiopathic Autism Autism 85qPCR ASD AU008403 AU0084 idiopathic Autism Autism 94 qPCR ASD AU008405AU0084 CNT qPCR AU016804 AU0168 CNT qPCR AU016805 AU0168 idiopathicAutism Autism 100 qPCR ASD AU028904 AU0289 CNT qPCR AU028905 AU0289idiopathic Autism Autism 110 qPCR ASD AU065603 AU0656 CNT qPCR AU065604AU0656 idiopathic Autism Autism 94 qPCR ASD AU0901301 AU0901 CNT qPCRAU0901302 AU0901 idiopathic Autism Autism 125 qPCR ASD AU1007301 AU1007CNT qPCR AU1007302 AU1007 idiopathic Autism Autism 119 qPCR ASDAU1054301 AU1054 idiopathic Autism Autism 90 qPCR ASD AU1054303 AU1054CNT qPCR AU1056301 AU1056 idiopathic Autism Autism 100 qPCR ASDAU1056303 AU1056 CNT qPCR AU1073301 AU1073 idiopathic Autism Autism 103qPCR ASD AU1073303 AU1073 CNT qPCR AU1193301 AU1193 CNT qPCR AU1193302AU1193 idiopathic Autism Autism 100 qPCR ASD AU1234301 AU1234 CNT qPCRAU1234302 AU1234 idiopathic Autism Autism 93 qPCR ASD AU1325301 AU1325idiopathic Autism Autism 100 qPCR ASD AU1325302 AU1325 CNT qPCRAU1327303 AU1327 CNT qPCR AU1327304 AU1327 idiopathic Autism Autism 114qPCR ASD AU1338303 AU1338 CNT qPCR AU1338304 AU1338 idiopathic AutismAutism 110 qPCR ASD AU1344302 AU1344 Idiopathic Autism Autism 128 qPCRASD AU1344303 AU1344 CNT qPCR AU1346302 AU1346 idiopathic Autism Autism125 qPCR ASD AU1346304 AU1346 CNT qPCR AU1412301 AU1412 idiopathicAutism Autism 131 qPCR ASD AU1412302 AU1412 CNT qPCR AU1424303 AU1424CNT qPCR AU1424304 AU1424 idiopathic Autism Autism 103 qPCR ASDAU1466301 AU1466 CNT qPCR AU1466302 AU1466 idiopathic Autism Autism 94qPCR ASD AU1549303 AU1549 idiopathic Autism Autism 125 qPCR ASDAU1549304 AU1549 CNT qPCR AU1562301 AU1562 CNT qPCR AU1562303 AU1562idiopathic Autism Autism 122 qPCR ASD AU1601302 AU1601 idiopathic AutismAutism 114 qPCR ASD AU1601303 AU1601 CNT qPCR AU1610304 AU1610 CNT qPCRAU1610306 AU1610 idiopathic Autism Autism 90 qPCR ASD AU039304 AU0393ASD with Autism microarray FMR1FM AU039305 AU0393 ASD with Autismmicroarray FMR1FM AU046703 AU0467 ASD with Autism microarray FMR1FMAU046706 AU0467 ASD with Autism microarray FMR1FM AU066703 AU0667 ASDwith Autism microarray FMR1FM AU066704 AU0667 ASD with Autism microarrayFMR1FM 01-19- ASD with not Autism Autism microarray dup15q 03-43- ASDwith Autism Autism microarray dup15q 02-7- ASD with Autism Autismmicroarray dup15q 98-19- ASD with Autism microarray dup15q AU006504AU0065 ASD with Autism microarray dup15q AU010603 AU0106 ASD with AutismSpectrum microarray dup15q AU010604 AU0106 ASD with Autism Autismmicroarray dup15q

TABLE 4 Gene networks identified by IPA using the 92 genes dysregulatedin ASD Genes in network Top functions Actin, ADAMTS4, ALOX5AP, ANG,CLEC2D, Cellular CTSS, CXCL11, dihydrotestosterone, Development ETV7,GLUL, GUSB, HLA-DOA, HLA-DQB1, Inflammatory HLX, IFNG, IL13, IL13RA1,IL13RA2, Disease IL4R, PTEN, RARA, RNF111, SKIL, SLC16A6, HematologicalSMURF2, STRA6, TGFB1, TGFBR1, System Development and Function TIMD4,TPM3, TRAM1, TYK2, UBE2D1, WWP1, XPO6 ANKRD29, ASNS, AZU1, BAMBI, BCAT1,BRP44, Cancer, CD70, DUSP5, DUSP16, EGF, FSTL1, GAS1, GFI1, GFI1B, GZMK,IL15, IL21R, Cell Death, MYC, MYL4, MYO6, NR3C1, PFKM, PLAGL1, PRDM1,RERE, RRM2, RRM2B, Cell Cycle SASH1, SOD2, STAT5B, TCF3, TERT, TP53,TYK2, ZFP36L1 ABCC5, AKR1C14, ALOX5AP, beta-estradiol, Cell Cycle,CCDC80, CCDC92, CEBPD, CLEC11A, CXADR, CXCL11, CXCR7, DBN1, DLG3, DUSP5,Cellular FLII, GCET2, GDA, IHPK2, IL1RN, Development, KRAS, MBTPS1,MED18, MED20, MED29, MMD, Hematological NBL1, NFkB, PCOLCE, PDGF BB,System Development and Function PRDX4, PSCD3, SLC35B1, SPCS2, TLR9, VAV2ACADSB, AGT, ANKRD25, CDC42, CDC25C, Cell Cycle, CDH3, CDKN2A, CLU,DPAGT1, EHMT1, ENTPD7, ERBB2, ERRFI1, GIMAP1, GTP, Histone Cancer, h3,HRAS, hydrogen peroxide, ITSN2, JAK1, JAKMIP1, LRP11, MCCC1, NUMB, Cellphosphate, RALBP1, RALGDS, Morphology RASGRF2, RASSF5, RFC1, RIN1,SLC7A11, SUZ12, TYK2, ZHX2

TABLE 5 ILLUSTRATIVE HUMAN DASD POLYNUCLEOTIDE SEQUENCES RETREIVED FROMGENBANK LIBRARY DATABASE USING THE DISCLOSURE IN TABLES 1-4 JAKMIP (SEQID NO: 7) GGGGGCTGCGCTCGCTACGTCCGCTGCTGCTGCCCGGCTCGGGCCTGAGCGCCGAGCAGGATCCCAAGTGATGGTGGTTTCCTCGGAGGGCGAGCTGAGTACTGCGCGACTGGTTAGCACGGTGGAGCTGGTAGCCACGCCTGCTGGCTGGCGTGCGTGAACAGGTGTGGACCGCAGGATCTCAGCACTCTGACCCAAGGGGAAGCATGTCGAAGAAAGGCCGGAGCAAGGGCGAGAAGCCCGAGATGGAGACGGACGCGGTGCAGATGGCCAACGAGGAGCTGCGGGCCAAGCTGACCAGCATTCAGATCGAGTTCCAGCAGGAAAAAAGCAAGGTGGGCAAACTGCGCGAGCGGCTGCAGGAGGCGAAGCTGGAGCGCGAGCAGGAGCAGCGACGGCACACGGCCTACATTTCGGAGCTCAAGGCCAAGCTGCATGAGGAGAAGACCAAGGAGCTGCAGGCGCTGCGCGAGGGGCTCATCCGGCAGCACGAGCAGGAGGCGGCGCGCACCGCCAAGATCAAGGAGGGCGAGCTGCAGCGGCTACAGGCCACGCTGAACGTGCTGCGCGACGGCGCGGCCGACAAGGTCAAGACGGCGCTGCTGACCGAGGCGCGCGAGGAGGCGCGCAGGGCCTTCGATGGAGAGCGCCTGCGGCTGCAGCAGGAGATCCTGGAGCTCAAGGCAGCGCGCAATCAGGCAGAGGAGGCGCTCAGTAACTGCATGCAGGCCGACAAGACCAAGGCAGCCGACCTGCGTGCCGCCTACCAGGCGCACCAAGACGAGGTGCACCGCATCAAGCGCGAGTGCGAGCGCGACATCCGCAGGCTGATGGATGAGATCAAAGGGAAAGACCGTGTGATTCTGGCCTTGGAGAAGGAACTTGGCGTGCAGGCTGGGCAGACCCAGAAGCTGCTTCTGCAGAAAGAGGCTTTGGATGAGCAGCTGGTTCAGGTCAAGGAGGCCGAGCGGCACCACAGTAGTCCAAAGAGAGAGCTCCCGCCCGGGATCGGGGACATGGTGGAGCTCATGGGCGTCCAGGATCAACATATGGACGAGCGAGATGTGAGGCGATTTCAACTAAAAATTGCTGAACTGAATTCAGTGATACGGAAGCTGGAAGACAGAAATACGCTGTTGGCAGATGAGAGGAATGAACTGCTGAAACGCTCACGAGAGACCGAGGTTCAGCTGAAGCCCCTGGTGGAGAAGAACAAGCGGATGAACAAGAAGAATGAGGATCTGTTGCAGAGTATCCAGAGGATGGAGGAGAAAATCAAGAACCTCACGCGGGAAAACGTGGAAATGAAAGAAAAGCTGTCAGCGCAGGCGTCTCTGAAGCGGCATACCTCCTTGAATGACCTCAGCCTGACGAGGGATGAGCAGGAGATCGAGTTCCTGAGGCTGCAGGTGCTGGAGCAGCAGCACGTCATTGACGACCTCTCACTGGAGAGAGAACGGCTGTTGCGCTCCAAAAGGCATCGAGGGAAAAGTCTGAAACCGCCCAAGAAGCATGTTGTGGAGACATTTTTTGGATTTGATGAGGAGTCTGTGGACTCAGAAACGTTGTCCGAAACATCCTACAACACAGACAGGACAGACAGGACCCCAGCCACGCCCGAAGAAGACTTGGACGATGCCACAGCCCGAGAGGAGGCTGACCTGCGCTTCTGCCAGCTGACCCGGGAGTACCAGGCCCTGCAACGCGCCTACGCCCTGCTCCAGGAGCAGGTGGGAGGCACGCTGGACGCTGAGAGGGAGGCCCGGACTCGGGAGCAGCTACAAGCTGATCTGCTGAGGTGTCAGGCCAAAATCGAAGATTTGGAGAAGTTACTGGTTGAGAAGGGACAGGATTCCAAGTGGGTTGAAGAGAAGCAGCTGCTCATCAGAACAAACCAAGACTTGCTGGAAAAGATTTACAGACTGGAAATGGAAGAGAACCAGCTGAAGAATGAAATGCAAGACGCCAAGGATCAGAACGAGCTGTTAGAATTCAGAGTGCTAGAACTCGAAGTAAGAGACTCTATCTGTTGTAAACTCTCAAACGGAGCAGACATTCTCTTTGAACCCAAACTGAAATTCATGTAAAGCTCTCAGATGTTTTCAAGCATGTGTAAAGGGGACATGTTATAGTTTCTTTCTTTCTTTCTTTCTTTTTTTTTTTAAATCTGTATGTTCAGAATAATTTCACTGCCTTAATGTGTTCTGGAGAGCGTGCTCACCCAAGTCTATGGACATGTACCAGAGCTAATATATTTATTGCCTATGGCTTGTTTTGCACTTAATAAAATAATTTGTTTT TACAAAAAAA STEAP (SEQID NO: 8) GCGGACGCGGGGCGCCAGCAGGTGGCGCTGGACGCGCAACGGACAAGGAGGCGGGGCCTGCAGCTGGCTTGGAGGCTCCGCGCTCTGGAGGCTCAGGCGCCGCGTGGGGCCCGCACCTCTGGGCAGCAGCGGCAGCCGAGACTCACGGTCAAGCTAAGGCGAAGAGTGGGTGGCTGAAGCCATACTATTTTATAGAATTAATGGAAAGCAGAAAAGACATCACAAACCAAGAAGAACTTTGGAAAATGAAGCCTAGGAGAAATTTAGAAGAAGACGATTATTTGCATAAGGACACGGGAGAGACCAGCATGCTAAAAAGACCTGTGCTTTTGCATTTGCACCAAACAGCCCATGCTGATGAATTTGACTGCCCTTCAGAACTTCAGCACACACAGGAACTCTTTCCACAGTGGCACTTGCCAATTAAAATAGCTGCTATTATAGCATCTCTGACTTTTCTTTACACTCTTCTGAGGGAAGTAATTCACCCTTTAGCAACTTCCCATCAACAATATTTTTATAAAATTCCAATCCTGGTCATCAACAAAGTCTTGCCAATGGTTTCCATCACTCTCTTGGCATTGGTTTACCTGCCAGGTGTGATAGCAGCAATTGTCCAACTTCATAATGGAACCAAGTATAAGAAGTTTCCACATTGGTTGGATAAGTGGATGTTAACAAGAAAGCAGTTTGGGCTTCTCAGTTTCTTTTTTGCTGTACTGCATGCAATTTATAGTCTGTCTTACCCAATGAGGCGATCCTACAGATACAAGTTGCTAAACTGGGCATATCAACAGGTCCAACAAAATAAAGAAGATGCCTGGATTGAGCATGATGTTTGGAGAATGGAGATTTATGTGTCTCTGGGAATTGTGGGATTGGCAATACTGGCTCTGTTGGCTGTGACATCTATTCCATCTGTGAGTGACTCTTTGACATGGAGAGAATTTCACTATATTCAGAGCAAGCTAGGAATTGTTTCCCTTCTACTGGGCACAATACACGCATTGATTTTTGCCTGGAATAAGTGGATAGATATAAAACAATTTGTATGGTATACACCTCCAACTTTTATGATAGCTGTTTTCCTTCCAATTGTTGTCCTGATATTTAAAAGCATACTATTCCTGCCATGCTTGAGGAAGAAGATACTGAAGATTAGACATGGTTGGGAAGACGTCACCAAAATTAACAAAACTGAGATATGTTCCCAGTTGTAGAATTACTGTTTACACACATTTTTGTTCAATATTGATATATTTTATCACCAACATTTCAAGTTTGTATTTGTTAATAAAAT GATTATTCAAGGAAAAAAAANAPRT (SEQ ID NO: 9) GCGGAGTCCGGACGTCGGGAGCAGGATGGCGGCGGAGCAGGACCCCGAGGCGCGCGCGGCGGCGCGGCCGCTGCTCACTGACCTCTACCAGGCCACCATGGCGTTGGGCTATTGGCGCGCGGGCCGGGCGCGGGACGCCGCCGAGTTCGAGCTCTTCTTCCGCCGCTGCCCGTTCGGCGGCGCCTTCGCCTTGGCCGCCGGCTTGCGCGACTGTGTGCGCTTCCTGCGCGCCTTCCGCCTGCGGGACGCCGACGTGCAGTTCCTGGCCTCGGTGCTGCCCCCAGACACGGATCCTGCGTTCTTCGAGCACCTTCGGGCCCTCGACTGCTCCGAGGTGACGGTGCGAGCCCTGCCCGAGGGCTCCCTCGCCTTCCCCGGAGTGCCGCTCCTGCAGGTGTCCGGGCCGCTCCTGGTGGTGCAGCTGCTGGAGACACCGCTGCTCTGCCTGGTCAGCTACGCCAGCCTGGTGGCCACCAACGCAGCGCGGCTTCGCTTGATCGCAGGGCCAGAGAAGCGGCTGCTAGAGATGGGCCTGAGGCGGGCTCAGGGCCCCGATGGGGGCCTGACAGCCTCCACCTACAGCTACCTGGGCGGCTTCGACAGCAGCAGCAACGTGCTAGCGGGCCAGCTGCGAGGTGTGCCGGTGGCCGGGACCCTGGCCCACTCCTTCGTCACTTCCTTTTCAGGCAGCGAGGTGCCCCCTGACCCGATGTTGGCGCCAGCAGCTGGTGAGGGCCCTGGGGTGGACCTGGCGGCCAAAGCCCAGGTGTGGCTGGAGCAGGTGTGTGCCCACCTGGGGCTGGGGGTGCAGGAGCCGCATCCAGGCGAGCGGGCAGCCTTTGTGGCCTATGCCTTGGCTTTTCCCCGGGCCTTCCAGGGCCTCCTGGACACCTACAGCGTGTGGAGGAGTGGTCTCCCCAACTTCCTAGCAGTCGCCCTGGCCCTGGGAGAGCTGGGCTACCGGGCAGTGGGCGTGAGGCTGGACAGTGGTGACCTGCTACAGCAGGCTCAGGAGATCCGCAAGGTCTTCCGAGCTGCTGCAGCCCAGTTCCAGGTGCCCTGGCTGGAGTCAGTCCTCATCGTAGTCAGCAACAACATTGACGAGGAGGCGCTGGCCCGACTGGCCCAGGAGGGCAGTGAGGTGAATGTCATTGGCATTGGCACCAGTGTGGTCACCTGCCCCCAACAGCCTTCCCTGGGTGGCGTCTATAAGCTGGTGGCCGTGGGGGGCCAGCCACGAATGAAGCTGACCGAGGACCCCGAGAAGCAGACGTTGCCTGGGAGCAAGGCTGCTTTCCGGCTCCTGGGCTCTGACGGGTCTCCACTCATGGACATGCTGCAGTTAGCAGAAGAGCCAGTGCCACAGGCTGGGCAGGAGCTGAGGGTGTGGCCTCCAGGGGCCCAGGAGCCCTGCACCGTGAGGCCAGCCCAGGTGGAGCCACTACTGCGGCTCTGCCTCCAGCAGGGACAGCTGTGTGAGCCGCTCCCATCCCTGGCAGAGTCTAGAGCCTTGGCCCAGCTGTCCCTGAGCCGACTCAGCCCTGAGCACAGGCGGCTGCGGAGCCCTGCACAGTACCAGGTGGTGCTGTCCGAGAGGCTGCAGGCCCTGGTGAACAGTCTGTGTGCGGGGCAGTCCCCCTGAGACTCGGAGCGGGGCTGACTGGAAACAACACGAATCACTCACTTTTCCCCACAAAAAA GABRA (SEQ ID NO: 10)GGGCTGGCTGAGCGCGGGCGAGTGTGAGCGCGAGTGTGCGCACGCCGCGGGAGCCTCTCTGCCCTCTCCTCGCACCCTGCTCAGGGCATCTGAAGAGCCTGGAAACGTGAACAGGCTTGAAGTATGGCATGTTGCAAAGATGGTTTCTGCCAAGAAGGTACCCGCGATCGCTCTGTCCGCCGGGGTCAGTTTCGCCCTCCTGCGCTTCCTGTGCCTGGCGGTTTGTTTAAACGAATCCCCAGGACAGAACCAAAAGGAGGAGAAATTGTGCACAGAAAATTTCACCCGCATCCTGGACAGTTTGCTCGATGGTTATGACAACAGGCTGCGTCCTGGATTTGGGGGTCCTGTTACAGAAGTGAAAACTGACATATATGTCACCAGCTTTGGACCTGTTTCTGATGTTGAAATGGAATACACAATGGATGTGTTCTTCAGGCAGACATGGATTGACAAAAGATTAAAATATGACGGCCCCATTGAAATTTTGAGATTGAACAATATGATGGTAACGAAAGTGTGGACCCCTGATACTTTCTTCAGGAATGGAAAGAAATCTGTCTCACATAATATGACAGCTCCAAATAAGCTTTTTAGAATTATGAGAAATGGTACTATTTTATACACAATGAGACTCACCATAAGTGCGGAGTGTCCCATGAGATTGGTGGATTTTCCCATGGATGGTCATGCATGCCCTTTGAAATTCGGGAGTTATGCCTATCCAAAGAGTGAGATGATCTATACCTGGACAAAAGGTCCTGAGAAATCAGTTGAAGTTCCGAAGGAGTCTTCCAGCTTAGTTCAATATGATTTGATTGGGCAAACCGTATCAAGTGAAACCATCAAATCAATTACGGGTGAATATATTGTTATGACGGTTTACTTCCACCTCAGACGGAAGATGGGTTATTTTATGATTCAGACCTATATTCCGTGCATTATGACAGTGATTCTTTCTCAAGTTTCATTTTGGATAAATAAAGAATCAGTTCCCGCTAGGACTGTATTTGGAATAACAACTGTCCTCACCATGACCACACTAAGCATCAGTGCACGACATTCTTTGCCCAAAGTGTCCTATGCTACCGCCATGGACTGGTTCATAGCTGTCTGCTTTGCTTTTGTATTTTCGGCCCTTATCGAGTTTGCTGCTGTCAACTATTTCACCAATATTCAAATGGAAAAAGCCAAAAGGAAGACATCAAAGCCCCCTCAGGAAGTTCCCGCTGCTCCAGTGCAGAGAGAGAAGCATCCTGAAGCCCCTCTGCAGAATACAAATGCCAATTTGAACATGAGAAAAAGAACAAATGCTTTGGTTCACTCTGAATCTGATGTTGGCAACAGAACTGAGGTGGGAAACCATTCAAGCAAATCTTCCACAGTTGTTCAAGAATCTTCTAAAGGCACACCTCGGTCTTACTTAGCTTCCAGTCCAAACCCATTCAGCCGTGCAAATGCAGCTGAAACCATATCTGCAGCAAGAGCACTTCCATCTGCTTCTCCTACTTCTATCCGAACTGGATATATGCCTCGAAAGGCTTCAGTTGGATCTGCTTCTACTCGTCACGTGTTTGGATCAAGACTGCAGAGGATAAAGACCACAGTTAATACCATAGGGGCTACTGGGAAGTTGTCAGCTACTCCTCCTCCATCGGCTCCACCACCTTCTGGATCTGGCACAAGTAAAATAGACAAATATGCCCGTATTCTCTTTCCAGTCACATTTGGGGCATTTAACATGGTTTATTGGGTTGTTTATTTATCTAAGGACACTATGGAGAAATCAGAAAGTCTAATGTAATTTCGTTGCTATAGTAGTTTGCTAAAAGATGATGAAAATGCAGAATGTCTTTTTAAATGTTTTTAAATATAAACAAATATTCTTTACTAAAATAAAAACTCTGTGTAATTTTTCCATTTAAAGATATAAGCCAGTTATTGGGAGAGTTAATTAATTCCTGAGTGAAAAAGTGAACTATGTTTTTTTTCAGAAAAATTATTTTAAAAGAACTCAGCATTCAGTTAGATAGAATACACACCATCCTGGAAAGTTGGGATAAGAGAAATAGAGCTATTAGAGACAAGTGGCGCATATTTTTTCATTGATATTTGAAAACAGACTATGACATTTTAAAAATCTGCCCTATGAGTATCAACCTGCCACCCTAAATTTCCCAGTGGCACTACCCTTAACCAGAATTGTTTATTAGATGTCATATGCAGTGACCTTTGGTGATCTTCTTAGGAACTTCAAGAAAAGGAATTTTCCTGTTAAATTAAACATTGGCAAAAGGAAATGGAATAGTATAAACACTGATCAATAGAGTAAAATATCTGCTGCATAAAAAACTAAGACAAAGACCAGAGGAAATATCTTCCCTTTCTTATGTTGGCTAAACAGTACTTAACAGTTGACTTGAAATTTTGTTCTCTGAGCCAAAGTTTAACTCATTGTATGAATTCTTTTTCATGGTAGTTCATTCAGTTATGTGTTTATTTACTACATAGTTATTCAGAGCCTACTGTGTTCCAGGAACTATGCTAGAAACTGTCTCTCTAGGAAAGCTCTTGCACCTTTATCTACAATATTACTTAAAAAGTAGAACAGTCAATGCATGCCAAAGAACCATAAACTAGCAGAGGACATTGCATTCTTTAGTGAAGGACATTTATTTAGAGTCTGACAACATATTCAAAATATTTTTCAGCCTCTACTGATAGTGGATAACAAATATATTTGTTCACATAACCACTTTGATGTCAGTACAACTTCAGCAATTGGTTTTCAAAATAGATGAGAATATGGTACAGATTGTTCTATAAGTGAAAAGCATTATGTACTTGAAAGTAAAAATCAGGGCAATATAAGACTTAATAGATTAACTGTCGCAAATTTGATCAGAGTCACAGAGTAGAATTTGATCAGAATCACAGAATCATCAGACATAGGAACTGAGCACAGGCTTTTTCAGGTGCTTTCCCCAAGATAGATCTAGATATTAGCTAGTGAAATGCTAAATTTTGAAGAGTTTTGTGTCCGTAGTTCTGTAATTCTGGGCAGTCATCATGTTGGTTTTTTTGGGAGTTTTTTTAAGGTTTAATAACTAAGGGGAATATTTTAAAATTAAGAGAGCAGCAAATGAAAGGAGTAAAGAAAAAAATAGCTGTCGGGTAGGATGCCACTGACTCTGCTATGTGATTTATCAGGGTTTTCATCTACTGACTTCTTTCTCATTAGGTAGGCTTAACAACTTACTTGAGAATTTTGCAACTGTCTATGCAGCTGAATCTAAGTATGGTTTTTTGTCATATTGCCTCTAGATTTTCTTCTGTGCTTCTCTCTATCTGCTCTGGAATGGATGAGTGAATGTGTTCTGGGTGTTTTAGGGAACATATTGATAGAAATGACCACCTTGCAGACAAAATCTCTCTCTCTCTTTTTGTTTTTATAGAAAAGAAGTGATAGTAGTTAATTGGCATCGATTTTTCAGATATTGCACTACCTTATAATGGTTGTTTTTGATACCTGAAAATTGTGCAAATGCCAAATATTAATTGTAAGTCATATCTGAGAAATCATTCTTGGCTGTCTTTCTAGGTATTCCATAGAATCAACACATTTTAAGGCTGAAAGATACTGTCGAAATCACCCAGTCCCAACCCCCCAATTTAAAATTGATTCTAGTAAAATTAGGTCCAGTAACACCTGTATATATGTATATATTTAATAAAGAGTACTTGTACAAAAAAGCTTATCATAAATTATTTCATGAATGTGAATAGATTTCTGGCCTTGAGGACGATGTTTGGAAATATGGTTGGAAGCACAATTCATTCGCTACTAGTTATTCAGAGCGTACTTTGTTCCAGGAACTATGCTGGAAACTGATAGTTCACTATTCTGATGAAAAGGCTGGTTTTGTTGTTGTTGTTGTTGTTGTTGTTGTTGTTTTTGGTGAGGAACTCTTACTCTTTGCTAATCTATTGGCCTATCTTAAGAAATAATTATTTGGTTATTCATTGGCTTCCTTTAAAAAAAAGTGTTCTTCTACAAATCTTACAGAGTGTAAAGAGATAAAAAGAAATGATTTTTTTTCTTACCTTTACATATGAAGGTTTAAAAATACATCATGTGGCCAAAGAATGAGAAAGGACAGAATTAACCAAGGATTCTTAATTGTTAATTTGAAGAACACATTAACCAGAAAGCTTAAACATTATTATTAAAATAAATTATTTCATTTGCACACAATGATAATAATGATGATAATAATAATAATAATAATAATAATAATAATAATAATAATAATATGTTGTTTGCTTTCCTTAACTGAATTTCATCAAATTCATTCAGTGATTTTTATTTTGGTCATTATTTCTATCTTCCCAATAACCCAGAGGTACATCAAGTAAAAACACTTTTGCAAAGAGCCAGTCACTTTTCCCTCTTAGAAAATCTCAGAAGAATGATTAGGGGCACCCAAAGTTCTGGTTATCTATGAATAATAATGAATAATTTTGGCTGAGAATGGTACCTTTATATAACTCTTTTAAAGATGAAATTCAGAATAATTTTATTCAGGACCTAGTATCTGCAGTCCATGTTGGCCACTAACTGCTACATAATTACTTTCAATTTCCTCCAGGCATGACTAGGTATAAACATATATTTACATGATTAATCCACATTTTAAAATGACTTGCACTTGTGCATATACATAGTACTTTGAATAATGTATATTTTAAATTGGACCTCACAAATCTTGTATTAAAGTAGGTAGAGAGTAGAGTCACACTTTTTTTGATGAAATGAGTCACAGAGAAGTTACATGACAAAGATAAAATCACAGGGTTGGAAAGAACTTTGAAAGGTCATTTTTCAATAATCTCACCTTAAAAGTGACCCCTTTTCAAACAAAGCATAAACAATTTATTTGTGGCTTATTTTACCCATGAGCTGGCCAATACTTCCTGGCCTAGAAAAATATCTCCACTCTCCCCATTCCTGTTCTTAATGGTCTTCTCTTCTCAATTTGTAAATATTCTCCAGTAAAAGACACCAGGTGTGGAGATGGGTCACTTAATTTCAAGTTCTGAGTGTCTTGTGGTTTTAAACAAGTCTCTTAACCTCTCTAAGACTGATTCCTCATTTATAACAGGGAGGAGGTGGATTATGTAATATCTAAGAGCTTTTCAGTTTTAATACTCTCTGATTCTCATAGTAAATACAAGCATACTCTTGTAAGAAAAACACTGAATACTTTGAACCACAAATAAGTGTTTATGGACAAAAATTTAGTTCTACTGCTTGATTACTTTAAGTTCATTGCTTGCTTCAAACTAAGGTTTCCCTACCTTCAAAAATATATATAACATCTCTCCATTTAGATAAAGTATATTCTTCCCCAGGGAAGACTGTATTCTTTAAAGACAGTAGAATATTGGCTATTTCTCTTTTCTGGGATTTCCTGGGCTTTTATTCACTAAATGGGCTCCTGCCCAGTTTGGGTACTGTGCTGATTTTTAGATATACAGAGATAAAAATAGCCCCTTTCCTTGCAGGTAGTCACAGTCCGGAAGAGATGATAGATGCATAGACAAAAAATTATGGGATGTAACTAATGCTTCAGTAGAATACAGCAGGGCCAAAACTAGAGTGATACTCATCTCAGGCACAAATATATATGGGGACATCAAAACTTCAATAATCAAGATACCTAATATTTAAGTGAATATTTTTAAAACTCAAAATTAATGCAAAAGATTTATGATGAACAGAATATACAAATTTTAAATAAATTAATAGAATCAGTAGTAATAATATTTTCTTCTGCCTCAGGCTCCAAAATTGCTCAGCATAGCACTGATCTAGATAGAGGTGGATAAAAGATCCACATAACTTAATTACATTGTTGAAGGAGAGGAACAAGAGTGATAGCAGAGAAGGTTTCACAGATATAATCTTAAGCTAAATTTTATAGAATGATTTGGAGTTCTTATAGCCTCCATTCCCACTTCCACAAACAAACAAAAAAATGAAAGCGCAATTTTATCAGCACCTGAGGCAGGAGAAGAGATGAGGATGTGGAAAGTCAAGGGGAACTCTTACTACAAATTCTTAGTTCTGTGTTTGGAGGTAAGGGTGAATGCAGGGAAGTGAGAGTTGTTGATGTTGGAAAGACCTAAAGAGCCTGGATTAGGAAGAGTTTTGTGTAAATTACTGAAGGAATGGAATTTGATCAGGATTAGTGGCAATTTATTAAAAATCTTGAAGAATAATACGATGAAAACTAACATCCTTATTTTTCTGTTTTAGAGAAAGCACCTGATTATTGTGTGAACTATGGTTTGGAGGGCCACAAATCTAGAGGTAGTGGAACAGTTGAGATCTCTTATTTTTTCTTTCCTGTCTTAAACTTTGCCTTCTATTCCTTTTTTCTTTTTTTGACCGCAATGACTTCATTGTCATTTGTAAAAGTGATACATGACTGTTAAAGATAGACATTGTAGAAACTTAAAAACAATCTCAAAATTCTACCACCCAGAAATCAGAAATATTTTGTATTTGGATATGTTTGTCTAGAAGAGTAGATTGCTTGATTAAAAGACAGAGTTTTATAAATGTAGAAGCCAATATGACATACATGCTGCCATTAAATAATAAAAAATTAAATTGACTTTTATCTCAAGATAAGATTAAAATTGGAGGGTAAAACTGAAGAAATTGTTGAAATTTGGATGGTGCTTCTTTAAAGCAAGAGATTTAGAGAGTCAGAGCTTTTTATCCCAGGTTTCAATCTTTATTTCAGGAAAATTTTCTGCAATTTTGTCTAAGTTAATTTTTTTTTTTTAAGAAATGCCAATTATGTCAAATCCCTTTTGTCTCTATACAATAGTCCCCAACTGTTTTGGCAACAGGCACGGGTGAGGGTGGGGGAATGGTTTTCAGATGAAATTGTTCCATCTCAGATCATCAGGCATTAGTCGATTCTCATAAGGAGCGGACAACCTTGCATGCCCTCCTATGAGAATCTAATGCCGCCGCTAATCTGACAGGAGGCAGAGCTCAGGCGATAATGCTCAATGGGTGCCACTCACCTCCTGTTGTGCTGCCCAATTCCTAACAGGTCATGGATAAGTACTGGTCAGTGGCCCAGGGGTTGGGGACCACTGCTCTGTAACTATTTACAGTATTCTTTGTAACTACTTTGGTATCTTTAAAATTTTCTTTAACTTTTGTCAACCATAACCCTAGTGAGTTTTCAGTTGTGATTATTCTATTTTGTAACTTCCAATGTACTTTTCAACTTCATAATGGTTAATTTTGTTTTCCATTGCTTTCTTGGTCACTGTCATTTCATTTTTCTTCTCTTTCTCTATTTTTACCATTGCTTTATTGAAATATCTTCTTTGAGCTTCTCATGTTTCTCTATTAATGAGATCATGTCTATAATATTTTTTGAGACTACAGAGACCTATTTATATAAACTCTTCCTCTATTTCTTGAAAAAGTTTTTATCTGGCGTGAGCTTCATCTGCTTTTTCATTGAGGTTTTATCTCCCTTGCCTATTTTTGGGGCTGGTTTCTTTCTATTAATCACTGAGCAGAGCCAGCTATTTACTGAACTACAGTGTGGGAAGTGGTGGGAGGGTGAGTCAGATCAGCCCACAGAAGCTATTTAAATTTCAGGTTTCAAGCCCACCTCCCCAAAACCGTTTTATGTGTTTTTTCTTCTGCCCAGGCACCATATTTTTATATCTATTTTTGACATTTGGGCGGCCTATGTAGTTCACAGTGTAAAACTCTCTGTTTTACTTTCTTTATGTTATTGCTAGTGATTATTGCTTGTAGTCCCACCCCCTTCCCCACATTAGCCTTAGTATTCTATATTATTCAGCAAGCTTGTTCACAACTCTCAAATATAGTCTATCAGAATTTTTATCTTTACCTCTACATTCCACTATTCGGAAGTATATAGTAAAATCGTATGAAGACAGATTTTGTCTTTTTCTTGCCTGTTTACACTCTATCTTACAGAGGTTTCAAACAAACCATCTTTGTTTGAAACATCGCAGGATAATGATACTTATTGAAATCTACATCCTGCCCAAGATATATGCCAGTCAATTTCCTTTCTCCTATTAGTGCAAATGGCTACCTACTTAAAAGCTGCTGACTATAGTTTGTCATCAATTACTTGTTAATTACAGATATGGTTTTCATTTTTTTTTCTCAATTTTTCATTATGTTATTTTTGGAGACTGTCTTAGGTGGGGAGTGAATTATAAATATTTTTATTTCACCACCTTTAACTGAGTCACCATATGTTATTTGCACTTCCAATAGACCAGGGATTGACCAGAGTATCTTTTACTGATATGACCACCAGAGCTACATGGCTTTTACCTTCTGTATATCAGATGTCACTGGAATCAAGCATTAAACCTAGATTAAATCTGGATTAAACCAGCTGTATGGCACCTTAGAAAATTGAGAAGGACTAGGAAGCTGATAGAAAGAACTCCTGTATAAAAAATAAATTCTTTTTACATTTTCCTTGGTAGTATTTCTGCAAGCTTCTTTAGTTTCTATAGGGGAATCCCAGAGTTTTCCTCTGAGATTGCTTCCCTTTCCTTTGTATTTTCATTTTTCCCTCTTGGGCTTTACTTTTCAGTCGTGTATTTCTATCCCAATGTTAATTTATTACACTGTTTATATTTTCCTGTCCTTCAATTCATCAGTAGATTAGTGAAGTACTTATTCCTTTAATTATAAGTATAAACGTTTTTAATTTCTTTTTGGATAACGATACATTTAATGGAAAATTTTTAAATGCTATGGTTTCTTAATAGCTTTTCTCTACCACTAATTACTTCCGTTAAAAAAAAAAAGAAATACCCATGCGTAATATAAAAGAAATTTTGAATAAAATGTTATCCTTTCCTTTTACTAGAGACAAACATCTCCACTTCAAAATGGAAAAATGGAACTATGCAAAGGAAATTTACGATATTCAAGAGTATGATATTATCACTGAACCGATTAATTTAATTGAAAATACTAACCTGCAAAAAAGAATTACACAAATGTATTTGAAATGTCATCAGATACCTCAAGCTATGACAGAGTTGATTATGTTACACAAGTAGTAAAGGATAAATTAAATATATTCTATATTAACACTAAGATTTAAAACTAATTATAGTCTTCTTTAATTTTTCTACTGTATACATTTCATCTAGTTTTCAGTAAAACAAACTTTTCATTACTTTTACTTATCCCATGCAAATCTAGTTGCTATAAGATATAACCTAATTTGGAAATACCTCCCTGTAATACATTGGAATTTTGGTGTGAGTGTGTGTGATCAGAGAGGATAAACAAATGGTGTCAGCAAAATAGAATGTAAAATATGGAGATAATGTGGATTTACACAATTTGATAAAAATTCTCCCTTGAATTTTGCAGATCATATATGAATATCATCATGTGAAGTGCAATTAGGATTTTTTCATATATTAATACATATACATCATCTCTTTAATAAAATATTTTGCTGAACTCGTTTAATATTTCTCATGTCTCATATTTCATATTTTCTCATATGTCTCATATTTTATATTTTCTCATATGTGTCTCATTTTTATTATTACTATAGTATCCATTAAAGGAGACTGGCAAATACCTGAACAAAGTTATGTTGTTGGCAATATAAGATATTACCAATTGCATAATATTATTTCCAAAGGCAAATTAATATGCAAACTATAGATTTCCATGGGTATCTATACAAATTATTACTTGAAGTCCATAGAAAACAGCTTCCTTAGAGGGAAGTAAAAGAAGCTCTGATAACAGACAATGTATAGCATTTCAGTTAAAGTTTGATTGACATATTTTTTTCTCAGTCTTTTATACTTGTGAATTATAGTCTACATTTTGTCTTTACAGATGTTGCTAACACAGTGCAGCATGTTGGCTTGTCATTTTACTGATTATGTATATTGTCTCCTAGTTCAAGTATACAGTAAATCAGAGCTTTCATTTTTCAAGGGGCAGAAAAATAATTGTTGGTTGATAAGGTAAGGTTATATGATTTTGAGGGAGCTTCATTGTAAATTGAATGAGAATCCGGTTTTCATGCAAAGGTGTATCTATGCGATAATACTTTGAATGTCTGTAGTTTGAAATAGAAGGTTAATTTTTTCCACTGGCTTATCCTTAATGAAGGAAATCCCCTGATAATTACTTTGATTCTGAAAATGTTGAAAACTCCAGAAGAAATAAAGCTTTTCTTTGATTTCTCAGCTGAAGTGTTGTACAAACTGAGGAATATGAAGTCCATTCCCTTCTTTCTTCTCTTGAATAATTTTAAAATTGTTTGTTTAGTTGTACAATTGAAAATATTCCTTATTATAGAAGTAAAAAAACAATAATGCATATTTCCTCATATCATAAAAACTAAATTTGTTATCATCTGAGCTTCAATATTCTTGCTCAATTAGAATAGTAAATATAAAGTGTGATATTTATAACAGTTCAAGGTTTGATAGGAGATAGAGGTGTTTTGATTTTATGGGAAAATTACTTCCTCAAGAACACATTTCTTGAGGTTTTAGTAATCACATTTGACTCCCTGAAATTGGCAATTTTATTCAATGTAGGAATATTATCATGGTATATTATAGTGAGGAGCACATAGTCATTATTATTTTGTTTTTGCAAATTTATTTTGAAAAAAAGGAGCATTATCAAATATTGGTTTACTATTTTAAAGTAATTCATCAGGAAATGATTTTTTAAACACTGTCCCTTTAAGTAAATGTCCCTTGCTTTTCAAAGCTCAACTTATTACTTATGGTAATAGAGTTACTTTGCTTCTTAAAACAAAGTTTATACTAGAGCATAGCATTGTAGAATTATTCAGGTCTCAATTTCTCACTGGAAAATGAGTCTTTAAAATAGTAAGGATTTAAATTTCTATAAAATTTAATCACAGATACTTATATTTTAAGATAAATGTGTTGGAGCTAAACTCTGGAATTATTAAAATATAAAACATTATATCCCTCTAATGTATTTTTTCTATTTAAATTTAAAACAATAAACATAAATATCTTTGGAAA GCET (SEQ ID NO: 11)CCACGCGTCCGGTGGTAAAGGGACGGAGGGGAAGCCCTGAGAGGACTGAGAGGATGGGAAATTCTCTGCTGAGAGAAAACAGGCGGCAGCAGAACACTCAAGAGATGCCTTGGAATGTGAGAATGCAAAGCCCCAAACAGAGAACATCCAGATGCTGGGATCACCATATCGCTGAAGGGTGTTTCTGCCTTCCATGGAAAAAAATACTCATTTTTGAAAAGAGGCAAGATTCCCAAAACGAAAATGAAAGAATGTCATCTACTCCCATCCAGGACAATGTTGACCAGACCTACTCAGAGGAGCTGTGCTATACCCTCATCAATCATCGGGTTCTCTGTACAAGGCCATCAGGGAACTCTGCTGAAGAGTACTATGAGAATGTTCCCTGCAAAGCTGAGAGACCCAGAGAGTCCTTGGGAGGAACTGAGACTGAGTATTCACTTCTACATATGCCTTCTACAGACCCCAGGCATGCCCGATCCCCAGAAGATGAATATGAACTTCTCATGCCTCACAGAATCTCCTCTCACTTTCTGCAACAGCCACGTCCACTTATGGCCCCTTCTGAGACTCAGTTTTCCCATTTATAGTGAAGTGGCTGGACTAGCATTTGTTTAGCACCAACAAATAAAAGGTGGGATGGGGGATCTGCCTGAAGCAGGGATGGGACACAAAGTCCCTCCAGCTTATCTCCCACAACAACCCTTTCCCTGCAGAGCATGGTTTGTATACCACAAGCCCTCTTAGCACGCAAAAGCCAAAATCTAAAGATCAACCATATCCTGAACAACACCATTTGAGAAAGAGGTAACCATCTTTGGTTCTACATGGTTTGGAGAGTATAGTGGTAGGAGGGGCTCCCTGATTCCCCTAAAGCTATGCACACCACAAGGGGCTCTGCTCTTCTGTCTGGGATCTTCTTATAAAGTGTTCCCATGATCATTCTCTAAAGTCACGAGGAAGCTTTACTCATCATACTAAGTGTGCCCAAGGGGGAGTTCACTCATTACTGTGACCTTCCAGCTCAGTCCCCACCCATGGGAGCCTGTGTTGCTCCTCTCACTCCATGTGTCTAAGTCATGTCTTTTACATAGTGTCCTTTGACCTGTTGGCCCCCATGGTCTGGTTAGTTATGTGAGTTGAATCAAGAGGCTCTAGGCCAGATGTTTACATAATTTTAACCTATATGATTTTATTTTTAACTTTGTATTTCTCCCTAGAAATCTTAATAAGACAATTATGCCATCAGACAATGTTAAGAAGAACGATCCTTGGAGATCCCGTAATCCCACTACCCTTCTTTGGCTCAGAGAGGATAATTTGCCTAATGATACATTAAAGTTAGTGGCAAAACTTAATTTGGAGCCTGATTTCCTACTGACTTCCAATTTAGTGCTCCCCCAGTATGCTAAATAGAAAGCCCTCTGCAATATATTAAATGTATACTAAATGTATATATTTAATAATGTCATGTATAAAATATGAATAAAATGTCCACAT AGGAAATTAACACATAAATIMD (SEQ ID NO: 12) ATAAGAGGTTGGGCTTTGGATAGATAGACAGACTCCTGGGTCCGGTCAACCGTCAAAATGTCCAAAGAACCTCTCATTCTCTGGCTGATGATTGAGTTTTGGTGGCTTTACCTGACACCAGTCACTTCAGAGACTGTTGTGACGGAGGTTTTGGGTCACCGGGTGACTTTGCCCTGTCTGTACTCATCCTGGTCTCACAACAGCAACAGCATGTGCTGGGGGAAAGACCAGTGCCCCTACTCCGGTTGCAAGGAGGCGCTCATCCGCACTGATGGAATGAGGGTGACCTCAAGAAAGTCAGCAAAATATAGACTTCAGGGGACTATCCCGAGAGGTGATGTCTCCTTGACCATCTTAAACCCCAGTGAAAGTGACAGCGGTGTGTACTGCTGCCGCATAGAAGTGCCTGGCTGGTTCAACGATGTAAAGATAAACGTGCGCCTGAATCTACAGAGAGCCTCAACAACCACGCACAGAACAGCAACCACCACCACACGCAGAACAACAACAACAAGCCCCACCACCACCCGACAAATGACAACAACCCCAGCTGCACTTCCAACAACAGTCGTGACCACACCCGATCTCACAACCGGAACACCACTCCAGATGACAACCATTGCCGTCTTCACAACAGCAAACACGTGCCTTTCACTAACCCCAAGCACCCTTCCGGAGGAAGCCACAGGTCTTCTGACTCCCGAGCCTTCTAAGGAAGGGCCCATCCTCACTGCAGAATCAGAAACTGTCCTCCCCAGTGATTCCTGGAGTAGTGCTGAGTCTACTTCTGCTGACACTGTCCTGCTGACATCCAAAGAGTCCAAAGTTTGGGATCTCCCATCAACATCCCACGTGTCAATGTGGAAAACGAGTGATTCTGTGTCTTCTCCTCAGCCTGGAGCATCTGATACAGCAGTTCCTGAGCAGAACAAAACAACAAAAACAGGACAGATGGATGGAATACCCATGTCAATGAAGAATGAAATGCCCATCTCCCAACTACTGATGATCATCGCCCCCTCCTTGGGATTTGTGCTCTTCGCATTGTTTGTGGCGTTTCTCCTGAGAGGGAAACTCATGGAAACCTATTGTTCGCAGAAACACACAAGGCTAGACTACATTGGAGATAGTAAAAATGTCCTCAATGACGTGCAGCATGGAAGGGAAGACGAAGACGGCCTTTTTACCCTCTAACAACGCAGTAGCATGTTAGATTGAGGATGGGGGCATGACACTCCAGTGTCAAAATAAGTCTTAGTAGATTTCCTTGTTTCATAAAAAAGACTCACTTAAAAAAAAA BAMBI (SEQ ID NO: 13)TTTACGGCGCGGAGCCGGAGAGACCTGGGCTGGCGCGGGCGGGAGCTGCGGCGGATACCCTTGCGTGCTGTGGAGACCCTACTCTCTTCGCTGAGAACGGCCGCTAGCGGGGACTGAAGGCCGGGAGCCCACTCCCGACCCGGGGCTAGCGTGCGTCCCTAGAGTCGAGCGGGGCAAGGGAGCCAGTGGCCGCCGACGGGGGACCGGGAAACTTTTCTGGGCTCCTGGGCGCGCCCTGTAGCCGCGCTCCATGCTCCGGCAGCGGCCCGAAACCCAGCCCCGCCGCTGACGGCGCCCGCCGCTCCGGGCAGGGCCCATGCCCTGCGCGCTCCGGGGGTCGTAGGCTGCCGCCGAGCCGGGGCTCCGGAAGCCGGCGGGGGCGCCGCGGCCGTGCGGGGCGTCAATGGATCGCCACTCCAGCTACATCTTCATCTGGCTGCAGCTGGAGCTCTGCGCCATGGCCGTGCTGCTCACCAAAGGTGAAATTCGATGCTACTGTGATGCTGCCCACTGTGTAGCCACTGGTTATATGTGTAAATCTGAGCTCAGCGCCTGCTTCTCTAGACTTCTTGATCCTCAGAACTCAAATTCCCCACTCACCCATGGCTGCCTGGACTCTCTTGCAAGCACGACAGACATCTGCCAAGCCAAACAGGCCCGAAACCACTCTGGCACCACCATACCCACATTGGAATGCTGTCATGAAGACATGTGCAATTACAGAGGGCTGCACGATGTTCTCTCTCCTCCCAGGGGTGAGGCCTCAGGACAAGGAAACAGGTATCAGCATGATGGTAGCAGAAACCTTATCACCAAGGTGCAGGAGCTGACTTCTTCCAAAGAGTTGTGGTTCCGGGCAGCGGTCATTGCCGTGCCCATTGCTGGAGGGCTGATTTTAGTGTTGCTTATTATGTTGGCCCTGAGGATGCTTCGAAGTGAAAATAAGAGGCTGCAGGATCAGCGGCAACAGATGCTCTCCCGTTTGCACTACAGCTTTCACGGACACCATTCCAAAAAGGGGCAGGTTGCAAAGTTAGACTTGGAATGCATGGTGCCGGTCAGTGGGCACGAGAACTGCTGTCTGACCTGTGATAAAATGAGACAAGCAGACCTCAGCAACGATAAGATCCTCTCGCTTGTTCACTGGGGCATGTACAGTGGGCACGGGAAGCTGGAATTCGTATGACGGAGTCTTATCTGAACTACACTTACTGAACAGCTTGAAGGCCTTTTGAGTTCTGCTGGACAGGAGCACTTTATCTGAAGACAAACTCATTTAATCATCTTTGAGAGACAAAATGACCTCTGCAAACAGAATCTTGGATATTTCTTCTGAAGGATTATTTGCACAGACTTAAATACAGTTAAATGTGTTATTTGCTTTTAAAATTATAAAAAGCAAAGAGAAGACTTTGTACACACTGTCACCAGGGTTATTTGCATCCAAGGGAGCTGGAATTGAGTACCTAAATAAACAAAAATGTGCCCTATGTAAGCTTCTACATCTTGATTTATTGTAAAGATTTAAAAGAAATATATATATTTTGTCTGAAATTTAATAGTGTCTTTCATAAATTTAACTGGGAAACGTGAGACAGTACATGTTAATTATACAAATGGCCATTTGCTGTTAATAATTTGTTCTCAACTCTAGGATGTGGCTTGGTTTTTTTTTTTCTCTTTTCTTTTTTAAACAAGACCAAGATCTTGCTTATTCTTCCATGAAAAAA SASH (SEQ ID NO: 14)ACGGCCATGGAGGACGCGGGAGCAGCTGGCCCGGGGCCGGAGCCTGAGCCCGAGCCCGAGCCGGAGCCCGAGCCCGCGCCGGAGCCGGAACCGGAGCCCAAGCCGGGTGCTGGCACATCCGAGGCGTTCTCCCGACTCTGGACCGACGTGATGGGTATCCTGGACGGTTCACTGGGAAACATCGATGACCTGGCGCAGCAGTATGCAGATTATTACAACACCTGTTTCTCCGACGTGTGCGAGAGGATGGAGGAGCTGCGGAAACGGCGGGTTTCCCAGGACCTGGAAGTGGAGAAACCCGATGCTAGCCCCACGTCACTTCAGCTGCGGTCCCAGATCGAAGAGTCGCTTGGCTTCTGTAGCGCCGTGTCAACCCCAGAAGTGGAAAGAAAGAACCCTCTTCATAAATCAAACTCAGAAGACAGCTCTGTAGGAAAAGGAGACTGGAAGAAGAAAAATAAGTATTTCTGGCAGAACTTCCGAAAGAACCAGAAAGGAATAATGAGACAGACTTCAAAAGGAGAAGACGTTGGTTATGTTGCCAGTGAAATAACGATGAGCGATGAGGAGCGGATTCAGCTAATGATGATGGTCAAAGAAAAGATGATCACAATTGAGGAAGCACTTGCTAGGCTCAAGGAATACGAGGCCCAGCACCGGCAGTCGGCTGCCCTGGACCCTGCTGACTGGCCAGATGGTTCTTACCCAACGTTTGATGGCTCATCAAACTGCAATTCAAGAGAACAATCGGATGATGAGACTGAGGAGTCGGTGAAGTTTAAGAGGTTACACAAGCTGGTAAACTCCACTCGCAGAGTCAGAAAGAAACTAATTAGGGTGGAAGAAATGAAAAAACCCAGCACTGAAGGTGGGGAGGAGCACGTGTTTGAGAATTCGCCGGTCCTGGATGAACGGTCCGCCCTCTACTCTGGCGTGCACAAGAAGCCCCTTTTCTTTGATGGCTCTCCTGAGAAACCTCCCGAAGATGACTCAGACTCTCTCACCACGTCTCCATCCTCCAGCAGCCTGGACACCTGGGGGGCTGGCCGGAAGTTGGTCAAAACCTTCAGCAAAGGAGAGAGCCGGGGCCTGATTAAGCCCCCCAAGAAGATGGGGACATTCTTCTCCTACCCAGAAGAAGAAAAGGCCCAGAAAGTGTCCCGCTCCCTCACCGAGGGGGAGATGAAGAAGGGTCTCGGGTCCCTAAGCCACGGGAGAACCTGCAGTTTTGGAGGATTTGACTTGACGAATCGCTCTCTGCACGTTGGCAGTAATAATTCTGACCCAATGGGTAAAGAAGGAGACTTTGTGTACAAAGAAGTCATCAAATCACCTACTGCCTCTCGCATCTCTCTTGGGAAAAAGGTGAAATCAGTGAAAGAGACGATGAGAAAGAGAATGTCTAAAAAATACAGCAGCTCTGTCTCTGAGCAGGACTCGGGCCTTGATGGAATGCCTGGCTCCCCTCCGCCTTCACAGCCCGACCCCGAACACTTGGACAAGCCCAAGCTCAAGGCCGGGGGTTCTGTAGAAAGTCTTCGCAGTTCTCTCAGTGGGCAGAGCTCCATGAGCGGTCAAACAGTGAGCACCACTGATTCCTCAACCAGCAACCGGGAAAGCGTCAAGTCGGAAGATGGGGATGACGAAGAGCCGCCTTACCGAGGCCCGTTCTGCGGGCGTGCCAGGGTGCACACCGACTTCACCCCCAGTCCCTATGACACAGACTCACTCAAGCTCAAGAAAGGAGATATCATCGATATAATCAGCAAGCCACCCATGGGGACCTGGATGGGCCTGCTGAACAACAAAGTCGGCACGTTCAAGTTCATCTACGTGGACGTGCTCAGTGAAGACGAGGAGAAACCCAAACGCCCCACCAGGAGGCGTCGGAAAGGACGACCACCCCAGCCCAAGTCTGTGGAGGATCTCCTGGATCGGATTAACCTAAAAGAGCACATGCCCACTTTCCTGTTCAATGGATATGAAGATTTGGACACCTTTAAGCTGCTGGAGGAGGAAGACTTGGATGAGTTAAATATCAGGGACCCGGAACACAGAGCTGTTCTCTTGACAGCAGTGGAGCTGTTACAAGAGTATGACAGTAACAGCGACCAGTCAGGATCCCAGGAGAAGCTGCTCGTTGACAGCCAGGGCCTGAGTGGATGCTCACCCCGAGACTCAGGATGCTACGAAAGCAGTGAGAACCTGGAAAACGGCAAGACTCGGAAAGCTAGCCTCCTATCTGCCAAGTCATCCACCGAGCCCAGCTTGAAGTCTTTTAGCAGAAACCAGTTGGGCAATTACCCAACATTGCCTTTAATGAAATCAGGGGATGCACTGAAGCAGGGACAGGAGGAGGGCAGGCTGGGTGGTGGCCTTGCCCCAGACACGTCCAAGAGCTGTGACCCACCTGGTGTGACTGGTTTGAATAAAAACCGAAGAAGCCTCCCAGTTTCCATCTGCCGGAGCTGTGAGACCCTGGAGGGCCCCCAGACTGTGGACACTTGGCCCCGATCCCATTCCCTGGATGACCTTCAAGTGGAGCCTGGTGCTGAGCAAGACGTGCCTACCGAGGTGACAGAACCGCCCCCTCAGATTGTACCTGAAGTGCCACAGAAGACGACCGCCTCTTCCACGAAGGCCCAGCCCCTGGAGCGAGACTCTGCTGTCGACAATGCATTGCTACTGACCCAAAGCAAGAGATTTTCTGAACCTCAGAAATTGACAACTAAGAAACTGGAGGGCTCAATCGCAGCCTCTGGTCGCGGCCTGTCACCCCCTCAGTGTTTGCCCAGAAACTATGATGCTCAGCCTCCTGGAGCTAAACACGGTTTAGCAAGGACGCCTCTGGAGGGCCACAGAAAAGGACACGAGTTTGAAGGAACACACCATCCCCTGGGCACCAAAGAAGGGGTAGATGCTGAGCAGAGAATGCAGCCCAAAATTCCATCACAGCCTCCACCTGTTCCTGCCAAAAAGAGCAGAGAACGCCTTGCTAACGGACTCCACCCTGTTCCCATGGGCCCCAGTGGGGCCCTCCCCAGTCCCGATGCGCCATGCCTGCCAGTGAAAAGGGGCAGCCCCGCCAGCCCCACCAGCCCTAGCGACTGTCCCCCAGCACTGGCTCCCAGGCCTCTCTCAGGGCAGGCGCCTGGCAGCCCACCAAGCACAAGGCCGCCCCCCTGGCTCTCAGAGCTCCCCGAGAACACAAGCCTCCAGGAGCACGGTGTGAAGCTGGGCCCGGCTTTGACCAGGAAGGTCTCCTGTGCCCGGGGAGTGGATCTAGAAACGCTCACTGAAAACAAGCTGCACGCTGAAGGCATCGATCTCACGGAGGAGCCGTATTCTGATAAGCATGGCCGCTGTGGGATTCCTGAAGCCCTGGTGCAGAGATACGCAGAGGACTTGGATCAGCCCGAGCGGGACGTCGCCGCCAACATGGACCAGATCCGGGTGAAGCAGCTTCGGAAGCAGCACCGCATGGCGATTCCAAGTGGTGGACTCACGGAAATCTGCCGAAAGCCCGTCTCTCCTGGGTGCATTTCGTCTGTGTCAGATTGGCTCATTTCCATCGGTCTGCCCATGTACGCCGGCACCCTCTCCACCGCGGGCTTCAGCACACTGAGCCAAGTGCCTTCTCTGTCTCACACTTGCCTTCAGGAGGCCGGCATCACAGAGGAGAGACACATAAGAAAGCTCCTATCTGCAGCCAGACTCTTCAAACTGCCGCCAGGCCCTGAGGCCATGTAGCCAGGCCCGGAATGGGCCTCTCTGGACAAGAGCCACCCTTTCACTGTGCATATGATGCTGATGCAATTCCTCCATCATCTCTGGACGTGCAGACCAGATCCAGAAGAAAGGCCTGGCGTGTGGCCAAACAGCGTGAAACCTTGGCACAGGACTGAGGATCCTCTCCTCCAGAAAAGCCCCCTCGAGGAAATAAATTAGTGCGGTTCTCTTTGACCTCCAAAGACAAGACAAGCACTTATTTTTATTTTCAGAAGACAAAAGAACCAAGATGCCAACTGGCTGCGAATGCTCTATCTCCAGTCTGTCTCTGTGTACTGGTAGAGGCTGGGAGGAGTAGGGGGCAGCCTGTTCCATTTCTGATAGTGCCCTTGCTCTTCTGTCTGTCATCTTGCAGGATGCCCGAGGGCCAGATGGGCTTAGCTAGGCCAAAGTAACAGACTCAAGAGTTATTGTACATTACTGACCACGCTCATTTGTTCAAAAGTTAGAACATCTGGCTGCACCAGGAAAAAAAAAAAAAAAAAGTCCTGTTCTTCTTTAGATAAACAAGAGACATTTTCATAATTGCTTTCTAGCAATCAGCTTTTATTTGCCTTAATATAAGCTTTTAAGCAGTTATCTAACTAGTGTCCACAACCCTGTAACCATACTTCCACATCTTCAGCTTAGGCAGACATCGAACCTCTCTGGGATGTTTCCAGCAAAAGTGAGCTTTTCTAATCGTCTCATTGTAACATGGCTTATTTTGTAGAGGTATTCATCAGCCACACACTTCATGTTGGTTTTTGGTTTTTAAGCTAACTACAAATCTAGTAAAAAGCTATCTGAAATTCACAAATATCATGTGTGTGCGTGCGTGCGTGCGCGTGTGTGTCTGTATTCATAGTGACTGCTTTTGGTTTTAACCAGTTTAGTATCGTTACTGTGTGGATCGTCGCGCTGCAGTATTGACTTGGAATCCTGACCATGTCCATCCCAAAATTCAGTCCTCAGTTAACGGATCATGTTTGCAAAAGGTCACTGTGAGGCTGCATATTTCAGAAAGATGTCCTTAATAAGGGAAGTCATGTATAAGATGTTTTCTAAAAGACTTTTCAGTATTACAACTAATACTATTATTATCCTTCTTTTTTTATTTAGATAATTCTTTTAATTTAAACAAAGGTTCACTATGGAACCAGACAAATCTCATTAGCCATGTGTTAAGTATTTGCTACTTTAAATTGTTTTACAACTGATTTCAGCACATTCTATCCTTTTTTTTTTTTGAAATGGAGTTTCGCTCTTGTCACCCAGGCTGGAGTGCAATGGCACGATCTTGGCTCACTGCAACCTCAGTCTCCCAGGTTCAAGTGATTCTCCTGCCTTAGCCTCCCGAGTAGCTGGGATTATAGGCACCCACCACCACGCCCAGCTAATTTTTGTATTATTAGTAGAGACAGGGTTTCACCATGTTGGCCAGGCTGGTCTCAAACTCAACTCCTGACCTCAGGTGGTCCACCCGCCTCAGCCTCCCAAAGTGCTGGGATTACAGGTGTGAGCCACCGCACCTGGCCTCTGTCCTCTTTTAGTCTAGTGTCTGGTTTTCTAGCAAACAGTAAATTTAAACAAGTAAACTATTATGGTTTCCATTGCTTACAAAATGATTTTCCTTTACATTCTTATCATGAACACTATTTTAAGCATCAAATGCAATCATCTAAAATATAAAGGTCAATCATTTATAATAGAAACACCTTGACCACAAGCCCTTGATTGAACATTTTATAATATTTCATCTACTTATTAAAACAAATAATTTCCCTTGGGTTGGAGGGGAAGTGATTTCATAAATTAATTAGAAAGCCATCTTTAGCATATTGCTTATGTCTGGATCCATGTTTCTGAGGAAAAAGACATTCTCAGGTGATGTATTTTTTTCATGCATTAGTATGCATTTTTAAAAAATAATGCATGTTTCTTTAATAATTAATTTTCATCTTCTATAAGATGCCATGTGAAGAAGTTGTGGAAATGTAGAATAAAAAGCTAAAGCTGCCAAATTTCTGTTGAACTCTTAAAAACAGCTCATGTTTGTTTGTCCTCTCGGGTTGTGGCCTAGCCTATTTGCAATGTAATGAAGCTGCAGGGTTCTTGTATAGCTAAAGCGTTCAATGCATTTCACGTGCTGTGGTGGATGTGGGTGCTGTAGACAGGCTTCTTCTCTTCCTGCTCTCAAAATACCTCGGCTTGACATTTGGACAGATCCTGTCATTGTTTAAGCTGAGCAAAAAACCACACAAAAGTTGTGTAAGAGATGAGATAACAAAGGAGCGAGAGAAATCTCATGTGAATTTCCAAGTTTTAATTCGTTCTCCATGAAGGATTTTCATTTCAGTGAAAGTCGCAGCAGAAGAGGGAACTTTCTGGAGTTTTTGAGAATGCCAAACCACATTTTTATCACACTTCTTTGGAAATCAATGCCTTTGCATAGAAAATCAAATTCAGGGACCACAAAGAATTTTCAGTGGGAATGTCTAGTCTGAGGGGTCTGAGGTTGTTTTTACTTTATTGTGTTGTTTAAATATTTTAAAAATATCTTTAGCGTTTGGTCTTTTTTTTTTCTGTAAACATTTAATTTGGTCTGAGAAAAGCTGAATGTTTGGGTGTGACGTTTGACTGAGGTGGATTGGGGCTGCCTGTGGACATTAGTGAACAGGTGGTAGGCTTCAGGAATATCCAGTTTTAATCAGTTGCATTTGGTACAGAATTTTGAGTAATGGTGAAAATTGTTGTCTTTGGAAAGCACAAAAGAAACCTGGAAAGGCAGTTCGGCTCAGGTAGCTACACATAACATTGTGTATGATTTTCACTTCAAAGCTGTCTGGAAGGAAATGCAGTCAGCTCCAGCTAGTACTATTTATGTACCCAGATAACTAAGATATTGTTTCATGGCCTTGCCTTAGTCAGAGGCCCTTTTCTCTGTCCTGAACCCCCAGGTATGGGTGAAATTGGAAATTACTAATCTATTGGAAATCAGTTCCTGACATAGTAAAGTTTGCTTTCATAACTGCAGCAAAAAAGGTCAACTTGCCAAGTCACTGCTGCCATGTGTGTACTGTATTATTTTCAGAAAAAAATATAATAGTCTGAGTCCAAGTTATCTTGATTTAAAATTGATAGAGAAAAGAAACTGTCGAGCAAGTTATATAACAACTAACAACATTGCACTTTCTGTATATGAAATCAATATTTAAATAACTTATTTTTCTCCATTGCTGTTCTTAAAAACATTGTAAGTAGCTGTAATATACCAGTACCAATATGTTCTTGCAATTGCTTCAGCCCAAGAAAGCTGTGTATTGTTTTAAAAATTGTAAAAATTATTGTGATGATTCATTTAGCATAAAGAGAGGTGGACGGAAGGGTTTTCCTATGTATCAAAACTTGTCTATAATTATGTCATCTATGTACCTAGAAAAAAGTAAATAAATTTCTTCAGTTGAATATG DACT (SEQ ID NO: 15)GGCGGTCGCGCGCAGGACTCGAGGGCTTCTAGCCACCGTCCCCGCCAGCGCCGCGCCCCGCCACAGGGCGGCATGAGCCCACCCGCGGCCGCAGCCCTAGCGCCCTGCTCCTCCGCCTGGGCGGCCCGGCTGCGGTGACGGCTCTCGCTGCCCGACTGGGGGCCATGAAGCCGAGTCCGGCCGGGACGGCGAAGGAGCTGGAGCCTCCGGCGCCGGCCCGAGGCGAGCAGCGCACGGCGGAGCCCGAGGGGCGCTGGCGGGAGAAGGGCGAGGCAGACACCGAGCGGCAGCGCACCCGGGAGCGGCAGGAGGCCACGCTGGCCGGGCTGGCGGAGCTGGAGTACCTGCGCCAGCGCCAAGAGCTGCTGGTCAGGGGCGCCCTGCGCGGCGCCGGGGGTGCGGGAGCCGCTGCGCCCCGCGCTGGGGAGCTACTGGGGGAGGCGGCGCAGCGCAGTCGCCTGGAGGAGAAGTTCTTGGAGGAGAACATCTTGCTGCTAAGAAAGCAATTGAACTGTTTGAGGCGAAGAGATGCTGGTTTGTTGAATCAGTTGCAAGAGCTTGACAAGCAGATAAGTGACCTGAGACTGGATGTAGAAAAGACATCTGAAGAGCACCTGGAGACAGACAGTCGGCCTAGCTCAGGGTTTTATGAGCTGAGTGATGGGGCTTCAGGATCCCTTTCCAATTCCTCTAACTCGGTGTTCAGTGAGTGTTTATCCAGTTGTCATTCCAGCACCTGCTTTTGCAGCCCCTTGGAGGCGACCTTGAGTCTCTCAGATGGTTGCCCCAAATCTGCAGATCTCATAGGATTGTTGGAATATAAAGAAGGCCACTGTGAAGACCAGGCCTCAGGGGCAGTTTGCCGTTCCCTCTCCACACCACAATTTAATTCCCTTGATGTCATTGCAGATGTGAATCCCAAGTACCAGTGTGATCTGGTGTCTAAAAACGGGAATGATGTATATCGCTATCCCAGTCCACTTCATGCTGTGGCTGTGCAGAGCCCAATGTTTCTCCTTTGTCTGACGGGCAACCCTCTGAGGGAAGAGGACAGGCTTGGAAACCATGCCAGTGACATTTGCGGTGGATCTGAGCTAGATGCCGTCAAAACAGACAGTTCCTTACCGTCCCCAAGCAGTCTGTGGTCTGCTTCCCATCCTTCATCCAGCAAGAAAATGGATGGCTACATTCTGAGCCTGGTCCAGAAAAAAACACACCCTGTAAGGACCAACAAACCAAGAACCAGCGTGAACGCTGACCCCACGAAAGGGCTTCTGAGGAACGGGAGCGTTTGTGTCAGAGCCCCGGGCGGTGTCTCACAGGGCAACAGTGTGAACCTTAAGAATTCGAAACAGGCGTGTCTGCCCTCTGGCGGGATACCTTCTCTGAACAATGGGACATTCTCCCCACCGAAGCAGTGGTCGAAAGAATCAAAGGCCGAACAAGCCGAAAGCAAGAGGGTGCCCCTGCCAGAGGGCTGCCCCTCAGGCGCTGCCTCCGACCTTCAGAGTAAGCACCTGCCAAAAACGGCCAAGCCAGCCTCGCAAGAACATGCTCGGTGTTCCGCCATTGGGACAGGGGAGTCCCCTAAGGAAAGCGCTCAGCTCTCAGGGGCCTCTCCAAAAGAGAGTCCTAGCAGAGGCCCTGCCCCGCCGCAGGAGAACAAAGTTGTACAGCCCCTGAAAAAGATGTCACAGAAAAACAGCCTGCAGGGCGTCCCCCCGGCCACTCCTCCCCTGCTGTCTACAGCTTTCCCCGTGGAAGAGAGGCCTGCCTTGGATTTCAAGAGCGAGGGCTCTTCCCAAAGCCTGGAGGAAGCGCACCTGGTCAAGGCCCAGTTTATCCCGGGGCAGCAGCCCAGTGTCAGGCTCCACCGGGGCCACAGGAACATGGGCGTCGTGAAGAACTCCAGCCTGAAGCACCGCGGCCCAGCCCTCCAGGGGCTGGAGAACGGCTTGCCCACCGTCAGGGAGAAAACGCGGGCCGGGAGCAAGAAGTGTCGCTTCCCAGATGACTTGGATACAAATAAGAAACTCAAGAAAGCCTCCTCCAAGGGGAGGAAGAGTGGGGGCGGGCCCGAGGCTGGTGTTCCCGGCAGGCCCGCGGGCGGGGGCCACAGGGCGGGGAGCAGGGCGCATGGCCACGGACGGGAGGCGGTGGTGGCCAAACCTAAGCACAAGCGAACTGACTACCGGCGGTGGAAGTCCTCGGCCGAGATTTCCTACGAAGAGGCCCTGAGGAGGGCCCGGCGCGGTCGCCGGGAGAATGTGGGGCTGTACCCCGCGCCTGTGCCTCTGCCCTACGCCAGCCCCTACGCCTACGTGGCTAGCGACTCCGAGTACTCGGCCGAGTGCGAGTCCCTGTTCCACTCCACCGTGGTGGACACCAGTGAGGACGAGCAGAGCAATTACACCACCAACTGCTTCGGGGACAGCGAGTCGAGTGTGAGCGAGGGCGAGTTCGTGGGGGAGAGCACAACCACCAGCGACTCTGAAGAAAGCGGGGGCTTAATTTGGTCCCAGTTTGTCCAGACTCTGCCCATTCAAACGGTAACGGCCCCAGACCTTCACAACCACCCCGCAAAAACCTTTGTCAAAATTAAGGCCTCACATAACCTCAAGAAGAAGATCCTCCGCTTTCGGTCTGGCTCTTTGAAACTGATGACGACGGTTTGAGTGACATCATTGGTGTAGAAAGTTTGTGTGTTTTTTTTTCTTCTCCCTAGTTGCCAAAATTAAAAAGGTGGTGTTTTCATTTTTGTATAATACTTTAATGGAATGCTTTTTAAAAAAATATAAAACCAAGGTAAATTATTGTTTCATCTTCACGTATGGATGCTAGTGCCTTTAATGGAAGGTAAAGAATGTTTTGCTAGTTAGAAGTACATATTGAGGTTTTAATGGTGGTGATAGTGAGTTTTGTGGCACCAGCTGTTTTTTATTTTAAACTTTCTGAGCATCCGGCAAGGTACAGGTTTTGATGTTCAAGTTTTATTGGGATAAGATCTTTTGATCCCAAGGTCAGGTGGATGGAATTTTTGGATTTATATTTGTTCCTTGAGTCTTCAGGGCAGTGTCTCCATGAGGGTTTTCCTGTTGAGGGGCACCACATACAATAGTGTGAAGTAGGTATGAGGGGCAGTCATTGTATTCTATAGTTTTTTTATGTAGTCTACATTTCTCAGATGTATCCCCATTCGGTTTTATTCTCAGAACTGTTACTAGACTCATGACTTGGAGGCCAAACCTTAAATCCAGAGATAGCAGCCTCGATAGGGACCTTAAAAGGATTCACAAAAACTTTTGCCACACTTGGTGCCTAGGCCCTGTTCCTAATAACCCCTTCTAGGGCCGTTTATCCAACATTTAGATGCCTTCTTTTCCCTCCCTAATTTGTAGCCAGTCCAACCTTTCATTCCTTGGAGGATTTAGTTTTGGGATAAAATTTTGGTCCTTGGGCACAGAGACATTCACTATTAATGAAGTAACCCTTGGGCATGACTCCAATCCCAGAATTGCTCACTGAGCGCTATGCCACCGAAGCGTTGACCTGAACATATTAGTGCAATCCAGTCCAGATTGGACCTTTGATCCTATGTGGAAGGGCTGTTTTTTAAGAAAAAATTTTTGGTAAACAGTATTGTGTAAAATTGCTTTTTGTATACCAATATATGCATGTTTTGTGCATGAGTAGTACTTGTGTTGATACTCCTGTTGATGTTAAATTACTATATAATATAAACAGTATGTGTTTTTATATATCATTGTGTAAATTTAATATAACATATGCAGTAATAAACCATTTGTTTTACTGCTGTTAAGTTTGTTATTTGGGTATAAAACCAGATGTTTACACCTGTAAAAAAAAAAAAAAAAAA DLG (SEQ ID NO: 16)GTGGAATCCGGCGTGGGCTGGGGGGTCCGAGCCGCGGGGGGCAGTGCCATGCACAAGCACCAGCACTGCTGTAAGTGCCCTGAGTGCTATGAGGTGACCCGCCTGGCCGCCCTGCGGCGCCTCGAGCCTCCGGGCTACGGCGACTGGCAAGTCCCCGACCCTTACGGGCCAGGTGGGGGCAACGGCGCCAGCGCGGGTTATGGGGGCTACAGCTCGCAGACCTTGCCCTCGCAGGCGGGGGCCACCCCCACCCCTCGCACCAAGGCCAAGCTCATCCCCACCGGCCGGGATGTGGGGCCGGTGCCTCCTAAGCCAGTCCCGGGCAAGAGCACCCCCAAACTCAACGGCAGCGGCCCCAGCTGGTGGCCAGAGTGCACCTGTACCAACCGGGACTGGTATGAGCAGGTGAATGGCAGTGATGGCATGTTCAAATATGAGGAAATCGTACTTGAGAGGGGCAACTCTGGCCTGGGCTTCAGTATCGCAGGTGGCATCGACAATCCCCATGTCCCTGATGACCCTGGCATCTTTATTACCAAGATTATCCCTGGTGGAGCAGCTGCCATGGATGGGAGGCTGGGGGTGAATGACTGTGTGCTGCGGGTGAATGAGGTGGACGTGTCGGAGGTGGTACACAGCCGGGCGGTGGAGGCGCTGAAGGAGGCAGGCCCTGTGGTGCGATTGGTGGTGCGGAGGCGACAGCCTCCACCCGAGACCATCATGGAGGTCAACCTGCTCAAAGGGCCCAAAGGCCTGGGTTTCAGCATTGCTGGGGGTATTGGCAACCAGCACATCCCAGGAGACAACAGCATCTACATCACCAAGATCATTGAGGGGGGTGCTGCTCAGAAGGATGGACGCCTACAGATTGGGGACCGGCTGCTGGCGGTGAACAACACCAATCTGCAGGATGTGAGGCACGAGGAAGCTGTGGCCTCACTGAAGAACACATCTGATATGGTGTATTTGAAGGTGGCCAAGCCAGGCAGCCTCCACCTCAACGACATGTACGCTCCCCCTGACTACGCCAGCACTTTTACTGCCTTGGCTGACAACCACATAAGCCATAATTCCAGCCTGGGTTATCTCGGGGCTGTGGAGAGCAAGGTCAGCTACCCTGCTCCTCCTCAGGTTCCCCCCACCCGCTACTCTCCTATTCCCAGGCACATGCTGGCTGAGGAGGACTTCACCAGAGAGCCTCGCAAGATCATCCTGCACAAAGGCTCCACAGGCCTGGGCTTCAACATCGTAGGAGGAGAGGATGGAGAAGGCATTTTTGTCTCCTTCATCCTGGCAGGAGGCCCAGCTGACCTGAGTGGGGAGCTGCGCAGGGGAGACCGGATCTTATCGGTGAATGGAGTGAATCTGAGGAATGCAACTCATGAGCAGGCTGCAGCTGCTCTGAAACGGGCCGGCCAGTCAGTCACCATTGTGGCCCAGTACAGACCTGAAGAATACAGTCGCTTTGAATCGAAGATACATGACTTACGAGAACAAATGATGAACAGCAGCATGAGCTCTGGGTCTGGGTCCCTCCGAACAAGTGAAAAGAGGTCCTTGTATGTCAGGGCCCTGTTTGATTATGATCGGACTCGGGACAGCTGCCTGCCAAGCCAGGGGCTCAGCTTCTCTTATGGTGACATTCTGCATGTCATTAATGCCTCTGATGATGAGTGGTGGCAGGCAAGGCTGGTGACCCCACACGGAGAAAGTGAGCAGATCGGTGTGATCCCCAGTAAGAAGAGGGTGGAAAAGAAAGAAAGAGCTCGATTGAAAACTGTGAAGTTCCATGCCAGGACGGGGATGATTGAGTCTAACAGGGACTTCCCGGGGTTAAGTGACGATTATTATGGAGCAAAGAACCTGAAAGGACAAGAGGATGCTATTTTGTCATATGAGCCAGTGACACGGCAAGAAATTCACTATGCAAGGCCTGTGATCATCCTGGGCCCAATGAAGGACCGAGTCAATGATGACCTGATCTCCGAATTTCCACATAAATTTGGATCCTGTGTGCCACATACTACCCGGCCTCGACGTGATAATGAGGTGGATGGACAAGACTACCACTTTGTGGTGTCCCGAGAACAAATGGAGAAAGATATTCAGGACAACAAGTTCATCGAGGCGGGCCAATTTAATGATAACCTCTATGGGACCAGCATCCAGTCAGTGCGGGCAGTTGCAGAGAGGGGCAAGCACTGCATCTTAGATGTTTCCGGCAATGCTATCAAGAGACTGCAGCAAGCACAACTTTACCCCATTGCCATTTTCATCAAGCCCAAGTCCATTGAAGCCCTTATGGAAATGAACCGAAGGCAGACATATGAACAAGCAAATAAGATCTATGACAAAGCCATGAAACTGGAGCAGGAATTTGGAGAGTACTTTACAGCCATTGTACAGGGTGACTCACTGGAAGAGATTTATAACAAAATCAAACAAATCATTGAGGACCAGTCTGGGCACTACATTTGGGTCCCATCCCCTGAAAAACTCTGAAGAATCCCCTCCAACCATTCTCTTGTGAACAGAAGAAATCAAGTCCCTCTTCCCTCCTCCCTCTTCATTCCTGTCCCCATG

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

1. A method of identifying a test mammalian cell having a gene expression profile observed in individuals diagnosed with autism comprising: observing an expression profile of at least one gene selected from the group consisting of SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 in the test mammalian cell; wherein an expression profile of a gene in the group that is at least two standard deviations from a mean expression profile of the gene in a control mammalian cell obtained from an individual not affected with autism identifies the test mammalian cell as having a gene expression profile observed in individuals diagnosed with autism.
 2. The method of claim 1, wherein the expression of a gene in the group is at least three, four or five standard deviations from the mean expression of the gene observed in a control mammalian cell.
 3. The method of claim 1, wherein the expression level of a gene in the group is at least 20, 30, 40, 50, 60 or 70% above or below the expression level of the gene observed in the control mammalian cell.
 4. The method of claim 1, wherein mRNA expression is observed.
 5. The method of claim 1, wherein polypeptide expression is observed.
 6. The method of claim 1, wherein the expression profile is observed using quantitative PCR (qPCR).
 7. The method of claim 1, wherein the expression profile is observed using Southern blotting.
 8. The method of claim 1, wherein the expression profile is observed using an antibody.
 9. The method of claim 1, wherein the expression profile of the test mammalian cell is observed using a microarray of polynucleotides.
 10. The method of claim 1, wherein the expression profile of the test mammalian cell is observed using a computer system comprising a processor element and a memory storage element adapted to process and store data from one or more expression profiles.
 11. The method of claim 1, wherein the test mammalian cell or the control mammalian cell is a leukocyte obtained from the peripheral blood.
 12. The method of claim 1, wherein the test mammalian cell is obtained from an individual previously identified as exhibiting restricted repetitive behaviors or speech delay.
 13. The method of claim 1, wherein the control mammalian cell is obtained from an individual previously identified as not exhibiting restricted repetitive behaviors or speech delay.
 14. The method of claim 1, wherein the test mammalian cell and the control mammalian cell are obtained from individuals who are related as siblings or as a parent and a child.
 15. The method of claim 14, wherein the control mammalian cell is obtained from a male sibling unaffected by autism.
 16. The method of claim 1, wherein the test mammalian cell is obtained from an individual identified as having a family member previously identified as exhibiting restricted repetitive behaviors or speech delay.
 17. The method of claim 1, wherein an expression profile of at least, 2, 3, 4, 5, 6, 7, 8, 9 or 10 genes in the group are observed. 